LPS-responsive chs1/beige-like anchor gene and therapeutic applications thereof

ABSTRACT

The present invention relates to a novel LPS-responsive and Beige-like Anchor gene (lrba), variants of the lrba gene, fragments of the lrba gene, and polypeptides encoded thereby. The subject invention also pertains to lrba interfering RNA, and uses thereof. In another aspect, the present invention also includes methods of inhibiting tumor growth in a patient by suppressing lrba function.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application is a National Stage filing of International ApplicationNo. PCT/US02/10350, filed Apr. 2, 2002, which claims the benefit ofprovisional patent application Ser. No. 60/280,107, filed Apr. 2, 2001,which is hereby incorporated by reference in its entirety, including allnucleic acid sequences, amino acid sequences, figures, tables, anddrawings.

The subject invention was made with government support under a researchproject supported by the National Institutes of Health Grant Nos. RO1DK54767, R21 AI44333, and PO1 NS27405. The government may have certainrights in this invention.

BACKGROUND OF THE INVENTION

Mutations in chs1/beige result in a deficiency in intracellulartransport of vesicles that leads to a generalized immune deficiency inmouse and man. The function of NK cells, CTL, and granulocytes isimpaired by these mutations indicating that polarized trafficking ofvesicles is controlled by chs1/beige proteins. However, a molecularexplanation for this defect has not been identified.

Lipopolysaccharide (LPS) is a potent inducer of maturation in B cells,monocytes, and dendritic cells that facilitates production ofinflammatory cytokines, nitric oxide, and antigen presentation so thatthese cells can participate in the immune response to bacterialpathogens (Harris, M. R. et al. Journal of Immunology, 1984, 133:1202;Tobias, P.S. et al. Progress in Clinical & Biol. Res., 1994, 388:31;Inazawa, M. et al. Lymphokine Res., 1985, 4:343). In an attempt toidentify genes involved in the maturation of immune cells, agene-trapping strategy was developed to identify mammalian genes whoseexpression is altered by cellular stimuli (Kerr, W. G. et al. ColdSpring Harbor Symposia on Quantitative Biology, 1989, 54:767). Severalnovel LPS-responsive genes were successfully trapped (Kerr, W. G. et al.Proc. Natl. Acad. of Sci. USA, 1996, 93:3947), including the SHIP genethat plays a role in controlling the maturation and proliferation of Bcells and monocytes/macrophages in vivo (Huber, M. et al. Prog. inBiophysics and Molecular Biol., 1999, 71:423; Ono, M. et al. Nature,1996, 383:263; Ono, M. et al. Cell, 1997, 90:293).

Chediak-Higashi Syndrome (CHS³) patients suffer from a systematic immunedeficiency characterized by a severe immune defect, hypopigmentation,progressive neurologic dysfunction and a bleeding diathesis (Spritz, R.A. Jour. of Clinical Immun., 1998, 18:97). Specific defects in immunecells include defects in T cell cytotoxicity (Abo, T. et al. Jour. ofClinical Investigation, 1982, 70:193; Baetz, K. et al. Jour. of Immun.,1995, 154:6122), killing by NK cells (Haliotis, T. et al. Jour. ofExper. Med., 1980, 151:1039), defective bactericidal activity andchemotaxis by granulocytes and monocytes (Clark, R. A. and H. R. KimballJour. of Clinical Investigation, 1971, 50:2645). CHS and beige lysosomesalso exhibit compartmental missorting of proteins (Takeuchi, K. et al.Jour. of Exper. Med., 1986, 163:665). Other studies have found thatbeige macrophages are defective for class II surface presentation(Faigle, W. et al. J. Cell Biol., 1998, 141:1121; Lem, L. et al. Jour.of Immun., 1999, 162:523) and that T cells in CHS patients are defectivefor CTLA4 surface expression (Barrat, F. J. et al. Proc. Natl. Acad. ofSci. USA, 1999, 96:8645). All cells in beige mice and CHS patients beargiant vesicles that cluster around the nucleus. Affected vesiclesinclude lysosomes, platelet dense granules, endosomes, and cytolyticgranules. These giant vesicles seem normal in several aspects except fortheir failure to release their contents, probably resulting frominability of the giant granules to mobilize and/or fuse with themembrane upon stimulation (Baetz, K. et al. Jour. of Immun., 1995,154:6122). However, despite these very provocative findings there stillremains no direct evidence that BG(beige)/CHS1 proteins associate withintracellular vesicles and thus a molecular explanation for defectivevesicle trafficking and protein missorting in these diseases is stillsought.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to a novel LPS-responsive and Beige-likeAnchor gene (lrba), its transcriptional/translational products, and thetargeting of the lrba gene for the treatment of cancer. Thus, thepresent application is directed to the lrba gene, variants of the lrbagene, fragments of the lrba gene, corresponding polypeptides encoding bysuch nucleotides, and uses thereof. The mouse lrba gene product isdisclosed herein in FIG. 1 and the human lrba gene product is disclosedherein in FIG. 9. The lrba gene is associated with the vesicular system,such as the Golgi complex, lysosomes, endoplasmic reticulum, plasmamembrane and perinuclear ER, and plays an important role in couplingsignal transduction and vesicle trafficking to enable polarizedsecretion and/or membrane deposition of immune effector molecules. Inone aspect, the lrba variants of the subject invention include fiveisoforms of the lrba gene, including lrba-α, lrba-β, lrba-δ, lrba-γ, andlrba-ε. The sequences of the mouse lrba cDNAs have been deposited inGENBANK with the following GENBANK accession numbers: lrba-α: AF187731,lrba-β: AF188506, lrba-γ AF188507.

The subject invention also relates to cloning and expression vectorscontaining the lrba gene, and fragments and variants thereof, and cellstransformed with such vectors.

In one aspect, the subject invention concerns lrba small interfering RNA(siRNA) sequences useful for the treatment of cancer. Preferably, thesiRNA duplex is formed by annealing single-stranded RNA sequences(ssRNA) of 5′CCAGCAAAGGUCUUGGCUAdTdT3′ (SEQ ID NO. 1) and5′CAGUCGGGUUUGCGACUGGdTdT3′ (SEQ ID NO. 2) from the lrba gene.

In a further aspect, the subject invention concerns methods ofinhibiting the growth of tumors in a patient by suppressing lrbafunction. According to the method of the subject invention, suppressionof lrba function can be carried out at various levels, including thelevels of gene transcription, translation, expression, orpost-expression. For example, suppression of lrba gene expression can becarried out using a variety of modalities known in the art forinterfering with the production of a functional product of a targetgene. For example, siRNA sequences, such as those described above, canbe administered to a patient in need thereof. The siRNA can be producedand administered exogenously, or the siRNA can be inserted into anappropriate vector and the vector can be administered to the patient forproduction of the siRNA in vivo, for example.

The subject invention also provides methods of detecting the presence oflrba nucleic acids, transcriptional products, or polypeptides in samplessuspected of containing lrba genes, transcriptional products, orpolypeptides.

Another aspect of the subject invention provides kits for detecting thepresence of lrba genes, lrba variants, lrba polypeptides, or lrbatranscriptional products obtained from the polynucleotide sequences.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A and 1B show the sequence and structure of the mouse lrba gene.FIG. 1A shows the predicted full-length amino acid sequence of thelrba-α (SEQ ID NO. 3) and lrba-β (SEQ ID NO. 4) (stopped at the boxed“R” with the additional sequence VSAVGSTLFLLLGSSK (SEQ ID NO. 5)) andlrba-γ (SEQ ID NO. 6) cDNAs (stopped at the boxed “I” with theadditional sequence GLPLLSLFAIH (SEQ ID NO. 7)). Bold amino acidsindicate the BEACH domain (2204-2482) based on alignment with 20 otherBEACH domains. Eight WD repeats predicted by an algorithm available athttp://bmerc-www.bu.edu/psa/request.htm, are underlined ordotted-underlined. The first three WD repeats are not predicted by otherprograms but resemble WD repeats and thus are referred to herein as WDL(WD-like) repeats. Two putative protein kinase A RII binding sites areshaded. The sequences of the mouse lrba cDNAs have been deposited inGENBANK with the following GENBANK accession numbers: lrba-α: AF187731,lrba-β: AF188506, lrba-γ. AF188507. FIG. 1B shows a schematic diagram ofmLRBA protein and alignment of the predicted mLRBA protein with itsorthologues and some paralogues. The stop sites for the lrba-β andlrba-γ are indicated by dashed lines. The human LRBA protein (SEQ ID NO.8) was predicted from a 9.9 kb “hybrid” cDNA sequence with the first 5′2577 nucleotides from this work (GENBANK accession numbers AF216648) andthe rest from the CDC4L partial cDNA sequence (GENBANK accession numbersM83822) (Feuchter, A. E. et al. (1992) Genomics 13:1237) except one Gwas added after position 5696 for two reasons: (i) the G base is presentin the cDNA sequence (GENBANK accession numbers AF217149); and (ii) thisaddition extended the CDC4L ORF by an additional 165 AA that had highhomology with mLRBA and other proteins shown in this figure. The dLRBAwas predicted from the drosophila melanogaster genomic sequence (GENBANKaccession number AE003433). cLRBA (GENBANK accession number T20719,Caenorhabditis elegans), aCDC4L (GENBANK accession number T00867,Arabidopsis thaliana), LSVA (GENBANK accession number AAD52096,Dictyostelium discoideum), hFAN (GENBANK accession number NP_(—)0035711,Homo sapiens), CHS1 (Chediak-Higashi Syndrome 1, GENBANK accessionnumber NP_(—)000072, Homo sapiens), mBG (GENBANK accession numberAAB60778, Mus musculus).

FIGS. 2A and 2B show the PKA binding sites in LRBA. In FIG. 2A, theconservation of hydrophobic amino acids of putative PKA binging sites inmLRBA (SEQ ID NO. 9), hLRBA (SEQ ID NO. 10), dLRBA (SEQ ID NOs. 11-12),and cLRBA (SEQ ID NO. 13) are shown by aligning with the known B1 and B2PKA RII tethering sites (underlined) in DAKAP550 (a partial cDNAsequence for dLRBA) along with other sequences in these regions. FIG. 2Bshows the predicted secondary structure of the putative PKA bindingsites in mLRBA (mLRBAb1, mLRBAb2). The hydrophobic amino acids on thehydrophobic side of the predicted amphipathic helices are boxed.

FIG. 3 shows the alignment of the C-terminal sequences of mLRBA (SEQ IDNO. 14), hLRBA (SEQ ID NO. 15), dLRBA (SEQ ID NO. 16), CHS1 (SEQ ID NO.17), and hFAN (SEQ ID NO. 18), which include the BEACH domains (in themiddle, boxed), 5 WD repeats and the 3 WDL repeats predicted in mLRBAand hLRBA. The predicted SH3, SH2 binding sites and tyrosine kinaserecognition sites are also boxed. The C-terminal difference of the threeisoforms of the mLRBA, α (SEQ ID NO. 14), β (SEQ ID NO. 19), and γ (SEQID NO. 20), are shown here (and FIG. 1B in more detail).

FIGS. 4A and 4B show that expression of lrba is inducible in B cells andmacrophages. FIG. 4A shows Northern blot hybridization of mRNA from Bcell line 70Z/3 and the macrophage cell line J774. Both cell lines werecultured with or without LPS for 20 hours. The poly A⁺RNA was purifiedfrom these cells, run on a denaturing formaldehyde agarose gell, andtransferred to a Hybond-N+ filter. The filter was hybridized with the2.5 kb probe that corresponds to the coding region of the lrba geneincluding the BEACH and WD domains, as described in the Materials andMethods section. The hybridized filter was exposed to X-ray film for 24hours. Similar amounts of β-actin mRNA were found in all mRNA tested(Actin panels). FIGS. 4B and 4C show expression of mRNA of three lrbaisoforms (α, β, and γ) in B cell lines (FIG. 4B) and tissues (FIG. 4C).Three isoform-specific primer pairs were used to detect the expressionof the three isoforms by RT-PCR, the expected product size of the RT-PCRproduct for the α form is 1344 bp, for the β form 836 bp, and for the γform 787 bp. Total RNA is analyzed. Aliquots (10 μl) of the PCR productswere resolved on 0.8% agarose gels. Three independent experiments wereperformed and yielded similar results.

FIGS. 5A-5I show subcellular localization of GFP-LRBA fusion proteinsrevealed by UV-fluorescence microscopy and laser-scan confocalmicroscopy. FIG. 5A shows the RAW 267.4 macrophage cell line (R7) stablytransfected with a BEACH-WD-GFP fusion construct. Most cells havediffuse, cytosolic GFP fluorescence, but some cells show vesicleassociation of the GFP fusion protein. In FIG. 5B, the same cell linefrom FIG. 5A was plated on glass-covered plates and stimulated with LPS(100 ng/ml) for 24 hours. Extensive vesicle association of the fusionprotein was observed. FIG. 5C shows RAW 267.4 macrophages stablytransfected with the control vector pEGFP-N2 that were cultured with 100ng/ml LPS stimulation. No obvious vesicle association of native GFP wasobserved. Magnification: 400×. FIG. 5D shows part of an R7 macrophagecell, showing GFP fluorescence. FIG. 5E shows the same part of an R7macrophage cell as in FIG. 5D, showing acidic lysosomes specificallylabeled by LysoTracker Red in living cells. FIG. 5F shows lysosomeco-localization (white part) of GFP fusion protein by overlappingpictures of FIGS. 5D and 5E; N=nucleus. FIG. 5G shows R7 macrophagecells, showing GFP fluorescence. FIG. 5H shows the same R7 macrophagecells as in FIG. 5G, showing prominent labeling of the Golgi complex(between the two nuclei) specifically labeled by BODIPY TR ceramide.Other intracellular membranes are weakly labeled. FIG. 5I shows Golgico-localization (white part) of GFP fusion protein by overlappingpictures shown in FIGS. 5G and 5H. Co-localization was determined byZeiss LSM 510 software, which allows for a reliability of 99% for actualpixels with both fluorophores. Co-localization mask pixels are convertedto white color for clarity. All cells were stimulated with LPS (100ng/ml) for 24 hours except for FIG. 5A.

FIGS. 6A-6F show immunoelectron microscopy of LRBA-GFP fusion protein.The LPS-stimulated R7 macrophage cells were fixed and processed forpostembedding immunocytochemistry. The cells were dehydrated andembedded in gelatin capsules in LR White resin. Ultrathin sections of LRWhite embedded cells were collected on nickel grids and immunolabeledwith rabbit-anti-GFP followed by labeling with anti-rabbit IgG-goldsecondary antibody, and finally stained with uranyl acetate and leadcitrate before examination with EM. FIG. 6A shows a clathrin-coated pit(endocytic, or coated vesicle) labeled with gold particles (open arrow).This is a vesicle forming on the cell surface. The fact that there isclathrin around this vacuole indicates that it is involved inendocytosis and not exocytosis. FIG. 6B shows intense labeling of aprimary lysosome (open arrow) and a vesicle on the cell surface (closedarrow). In FIG. 6C, the black arrows show ribosomes lining a profile ofendoplasmic reticulum (er). There are three gold particles labeling theER (open arrow). The gray structure next to the ER is a mitochondrion(m), which is not labeled. FIG. 6D shows a Golgi region of a celllabeled for GFP. The open arrows show gold particles on a Golgicisterna. FIG. 6E shows labeling of endoplasmic reticulum comprising theperinuclear cisterna (open arrows), and labeling of the plasma membraneof the cell (closed arrows). FIG. 6F shows gold particles surrounding asecondary lysosome in a cell (*). At the top of the lysosome is a coatedvesicle (closed arrow) fusing with the lysosome. A portion of ERsurrounds the bottom of the lysosome, which is also labeled with goldparticles (open arrow). Labeling of the perimeter of the secondarylysosome shows routing of GFP from the cell surface to the lysosomelimiting membrane. In FIGS. 6A-6F, e=extracellular space; n=nucleus;er=endoplasmic reticulum; g=Golgi; m=mitochondrion; c=cytoplasm. Thesize of gold particles is 10 nm.

FIG. 7 shows a model of vesicle secretion for WBW protein family usingthe lrba gene as a prototype. Following immune cell activation, theBEACH domain binds to vesicles containing cargo proteins and membraneproteins for secretion or deposition in the plasma membrane. The anchordomain binds to microtubules to move the vesicles to the membrane wherethe WD domain binds to phosphorylated sequences of membrane receptorcomplexes to mediate the fusion of the vesicles with the membrane, thusreleasing the cargo proteins or depositing membrane proteins on theplasma membrane of immune cells.

FIG. 8 shows a Western blot of a Raw 264.7 macrophage cell line andstably transfected Raw 264.7 cell lines, demonstrating inhibition ofapoptosis by LRBA fusion proteins. 586-2 cells were transfected withBEACH-GFP construct; R7 cells were transfected with BEACH-WD-GFPconstruct; and RGFP cells were transfected with pEGFP vector. The levelof both cleaved PARP (poly(ADP-ribose) polymerase and cleaved caspase 3are higher in control cell lines (Raw 264.7 and RGFP) than in LRBAtransfected Raw 264.7 cell lines (586-2 and R7), suggesting LRBAconstructs can prevent cells from apoptosis induced by staursporine.

FIG. 9 shows the predicted full-length amino acid sequence and structureof the human LRBA gene and its five isoforms (SEQ ID NO:182). Eachisoform is shown by α (SEQ ID NO. 8), β (SEQ ID NO. 21), γ (SEQ ID NO.22), δ (SEQ ID NO. 23), ε (SEQ ID NO. 24) at the right of eachC-terminus or the five amino acid insertion(γ). Residues in italicletters indicate isoform-specific sequences. Asterisk *=stop codon.Sequences are connected by arrows. The numbers at the right are for theα form. The domains are shaded and named above each domain. Five WDrepeats predicted by an algorithm available on the protein sequenceanalysis (PSA) server at the Boston University website are also shadedor boxed. HSH (helix-sheet-helix); SET: Rich in Serine(S), Glutamicacid(E) and Threonine(T). G peptide has five consecutive glycine. Thetwo potential start codons are boxed. The sequences of the LRBA cDNAshave been deposited in GenBank (accession number NM_(—)006726).

FIG. 10 shows secondary structure prediction and alignment of the HSHdomain in several WBW proteins (SEQ ID NOs. 25-31). Sequence positionshighlighted in magenta and yellow correspond, respectively, to helicesand strands. Sequence positions highlighted in blue are potentialglycosylation sites. Squared positions correspond to conserved residuesfound in the three WBW protein. The positions of the predicted helicalregions of the HSH structure are indicated as tubes at the top of thesequences. Sequences having homologues in FIG. 9 were analyzed asmultiple sequence alignments using the Jpred² method (Cuff, J. A. et al.(1998) Bioinformatics 14:892-893; Cuff J. A. and Barton, G. J. (1999)Proteins: Structure, Function and Genetics 34:508-519; Cuff, J. A. andBarton, G. J. (1999) Proteins: Structure, Function and Genetics40:502-511). Several sequences that, after a first prediction run, werefound to have more than 25% homology in one of the three conservedhelical regions were reprocessed together as a multiple sequencealignment using Jpred² to refine the prediction of that particularregion. Secondary structure predictions were performed by the Jpred²method. Rectangles indicate -helices and arrows indicate -strands. HSH(helix-sheet-helix) domain: Several WBW proteins have a high homologyand a common predicted protein secondary structure (HSH structure) overan 100 amino acid stretch near their N-terminus, as shown in FIG. 10.Because the HSH domain exists in evolutionarily very distant species(Dictyostelium is a cellular slime mold, more ancient than yeast), itmay have important function in a cell's life. SET domain: rich in serine(S, 13.70%), glutamic acid (E, 13.40%) and threonine (T, 9.03%). Itsfunction is still unknown. This domain is very hydrophobic and has avery high antigenic index. PI is 3.96.

FIG. 11 shows the genomic structure of the human LRBA gene. The genecontains 59 exons, which span more than 700 kb. The exon/intronstructure of the LRBA gene is mapped to the corresponding cDNA regionsencoded by each exon. Location and size of exons and introns are drawnto scale (GenBank accession number NM_(—)006726).

FIG. 12 shows a molecular phylogenic tree of the amino acid sequences ofWBW genes from various species. The tree was constructed by the neighborjoining method, based on sequence alignment conducted by CLUSTALXsoftware using either whole length sequence or only the BEACH domain,which gave very similar results. This indicates that the BEACH domain isco-evolving with the rest sequence of the gene and, as the wholesequences of some WBW genes are still unavailable (moreover, the lengthof the BEACH domain is relatively consistent (around 278 amino acids)),using the BEACH domain seems more reasonable. Thus, FIG. 12 is based onthe BEACH domain. All the sequences are from GeneBank. The numbers inparenthesis are GI numbers.

FIG. 13 shows alternative splicing of the human LRBA gene. The solid orgray box indicates coding exon, and the hatched box indicates UTR(untranslated region). The top numbers indicate exons in the main form(constitutive isoform versus alternative isoform) of human lrba, whilethe bottom numbers indicate alternative splicing isoforms of the humanLRBA gene. The single Greek letters denote the five isoforms. The LRBAδhas a 310 bp Alu sequence at its poly(A) tail. 5′-1, 3′-1 and 3′-2indicate 5′ end and 3′ end splicing, while I-1 and I-2 representinternal splicing. 5′-1 splicing gives alternative transcription startsite and suggests alternative promoter for human LRBA gene. The internalsplicing I-1 interrupts the coding sequence of LRBA, splitting LRBA intotwo open reading frames (ORF), and thus alternative potential startcodon ATG (the meaning of this splicing is further described anddiscussed later). Another internal alternative splicing I-2 is a 15 bpsequence in frame with the main ORF, inserting a YLLLQ (SEQ ID NO. 32)insertion into the human LRBA protein (noting that the l and w arehydrophobic amino acids). AATAAA indicate a polyadenine signal. 3′-1 and3′-2 splicing generate two additional different 3′ UTR tails for humanLRBA gene. The isoform identification was conducted by using thefollowing cultured cells and tissues: (1.) human pre-B (6417) cells;(2.) human Raji B cells; (3.) 293 cells; (4.) human MCF7 breast cancercells; (5.) human HTB4 lung cancer; (6.) human H322 human lung cancer;(7.) human A539 human lung cancer; (8.) human lung carcinoma; (9.) humanlung carcinoma adjacent tissue; (10.) human B-cell lymphoma; (11.) humanB-cell lymphoma; and (12.) normal adjacent tissue (3 pairs of tumortissue and adjacent tissue of human prostate).

FIG. 14 shows results of a 5′RACE (rapid amplification of cDNA end)procedure and 3′RACE procedure, respectively, conducted on the humanlrba gene. In FIG. 14, the lower band contains an AluSx repeat sequence312 bp long. RNAs were from: (1.) pre-B (6417); (2.) Raji B cells; (3.)293 cells; (4.) MCF7 breast cells; (5.) HTB4 lung cancer; (6.) H322human lung cancer; (7.) A539 human lung cancer; (8.) human lungcarcinoma; (9.) human lung carcinoma adjacent tissue; (10.) B-celllymphoma; (11.) B-cell lymphoma; and (12.) normal adjacent tissue.

FIG. 15 shows the 5′ end of the human lrbaε isoform with a long 5′ UTR(SEQ ID NO. 33). There are four small ORFs before the major ORF of thehuman lrba gene. The longest small ORF encodes the first 73 amino acidsof the hlrba protein (SEQ ID NO. 34) and is in frame with the major ORF,though there are four in-frame stop codons and 6 out-of-frame stopcodons, in between which would prevent potential read-through that makesa fusion protein. The other three ORFs encode 20 amino acids, 18 aminoacids, and 15 amino acids, respectively. The partial major codingsequence is in bold (SEQ ID NO. 35). The amino acid sequence in italicsis present in the main form of the LRBA gene but absent in the deltaform of the LRBA gene (SEQ ID NO. 36). The grey shaded sequence is theextra exon that has interrupted the LRBA sequence.

FIG. 16 shows the predicted secondary structure of RNA sequence betweenthe two ORF of human lrbaδ (SEQ ID NO:181). The free energy for thestructure is −40.29 kcal/mol. This suggests a potential IRES (internalribosome entry signal). There is no homologous sequence between IRES,however they all have complex secondary structure like long stemstructure.

FIG. 17 shows the promoter and part of the 5′ cDNA sequence of the humanlrba gene (SEQ ID NO. 37). Transcription start sites as determined by5′RACE procedure are indicated by arrows. Sequence for a CpG island isin bold. The DNA consensus binding motifs for various transcriptionfactors shown in the region −1561 to +1 were identified using theTFSEARCH (version 1.3) software (Yukata Akiyama (Kyoto Univ.)), thefirst nucleotide of the most 5′ cDNA denoted as 1. The initiatormethionine is in bold. The transcription binding sites are shaded,boxed, or underlined. The genomic sequences have GenBank accessionnumber AC104796.

FIGS. 18A and 18B show RT-PCR of human prostate tumor tissue andadjacent normal tissue, demonstrating that LRBA expression is increasedin human prostate cancer relative to matched normal tissue controls.FIG. 18A shows RT-PCR detection of human LRBA mRNA. FIG. 18B showsRT-PCR detection of human β-Actin mRNA to control for the amounts ofmRNA present. The PCR cycle parameters were as follows: 94° C. for 30seconds, 68° C. for 30 seconds, 72° C. for 1 minute, 25 cycles. Thesources from the matched samples are (from left to right) 1, 3, and 5:prostate adenocarcinoma tissue; 2, 4, and 6: normal prostate tissue.Samples 1 & 2, 3 & 4, and 5 & 6, are matched pairs from three differentprostate cancer patients.

FIG. 19 shows growth inhibition of human breast cancer cells byexpression of a dominant negative human LRBA mutant. MCF7 human breastcancer cells were seeded (1×10⁴/well) into a 96-well plate. On thesecond day, cells were infected with various titers of a recombinantadenovirus that contains a dominant negative LRBA mutant, in thepresence or absence of doxycycline. The BW-GFP mutant comprises theBEACH and WD domains of LRBA fused to GFP. The adenoviral vector has atetracycline-responsive promoter that is repressed in the presence ofdoxycycline and, thus, the BW-GFP mutant is expressed in the absence ofdoxycycline. Three days post-infection, the cells were labeled with³H-thymidine, the cells harvested and CPM incorporated into highmolecular weight DNA counted as a measure of cell proliferation (DNAsynthesis).

FIGS. 20A-20C show the knock-down of Lrba expression by LRBA siRNAtreatment and death of cancer cells. HeLa cells were plated 2×10⁴cells/well of a 24-well dish. The next day, cells were transfected asindicated or were left untreated (Blank). The cells were photographed 72hours after transfected and the wells harvested for cell counting. HeLacells (human adenocarcinoma) transfected with Lrba siRNA andlipofectamine (FIG. 20A) or mock transfected with H₂O and lipofectamine(FIG. 20B). Magnification is 400×. Note the presence of apoptotic ornecrotic cell bodies as well as the spindly, stressed morphology of theremaining adherent cells in the siRNA Lrba-treated well. FIG. 20C showsabsolute cell numbers recovered as determined by Coulter Counter.Students' T-test: P<0.0006 for mock versus Lrba siRNA; P<0.0036 forBlank versus Lrba siRNA; P<0.2271 for mock versus blank. The siRNAtreated cultures show a statistically significant decrease in cellnumber as compared to either mock or blank cultures, but there is nosignificant difference in the number of cells recovered from the mockand blank cultures. The RNA sequences that were annealed to make theLrba siRNA were: Lrba sense-strand: 5′CCAGCAAAGGUCUUGGCUAdTdT3′ (SEQ IDNO. 1); Lrba antisense-strand: 5′UAGCCAAGACCUUUGCUGGdTdT3′ (SEQ ID NO.38).

FIGS. 21A-21D show silencing of the Lrba gene in MCF7 human breastcancer cells and MCF10A human breast normal cells by two pairs of LrbasiRNA (siRNA1 and siRNA2), demonstrating that Lrba siRNAs selectivelykill human breast cancer cells but not normal cells. MCF7 cells (FIG.21A-21C) and MCF10A cells (FIG. 21D) were seeded at 2×10⁴ cells per wellin 24-well plates. One day later, the cells were transfected with LrbasiRNAs or with scramble siRNA as a negative control usingoligofectamine. After 72 hours of siRNA treatment, the photos (FIG. 21A,MCF7 transfected with siRNA1; FIG. 21B, MCF7 transfected with scramblesiRNA negative control; magnification 400×) were taken and the cellnumbers were counted by a Coulter counter. T-test: FIG. 21C (MCF7),P=0.0009 for scramble negative control versus siRNA1; P=0.0005 forscramble negative control 1 versus Lrba siRNA2; P=0.004 for siRNA1versus siRNA2. FIG. 21D (MCF10A), P=0.4070 for scramble negative controlversus siRNA1; P=0.9456 for scramble negative control 1 versus LrbasiRNA2; P=0.0514 for siRNA1 versus siRNA2. The siRNA sequences: siRNA1:CCAGCAAAGGUCUUGGCUAdTdT (SEQ ID NO. 1); siRNA2: GGGCACUCUUUCUGUCACCdTdT(SEQ ID NO. 39); scramble negative control: CAGUCGGGUUUGCGACUGGdTdT (SEQID NO. 2).

FIGS. 22A-22F show upregulation of lrba promoter activity by p53 and E2Ftranscription factors. The GFP reporter (GFP gene is placed downstreamof the lrba gene promoter, designated pLP-GFP) construct was transfectedinto 293T cells with or without p53 or E2F wild type vector. Thepictures were taken one day after transfection. FACS analysis wascarried out 60 hours after transfection. The results show that there is0.7% GFP positive cells in pLP-GFP only (FIGS. 22A and 22D), 6.88% inpLP-GFP+p53 vector (FIGS. 22B and 22E), 2.06% in pLP-GFP+pE2F1 vector(FIGS. 22C and 22F), suggesting that only a small fraction of cells havedetectable lrba promoter activity, p53 and E2F can induce the lrbapromoter activity to 9.8, 3-fold respectively. p53 and E2F are importantcell cycle and apoptosis mediators. All or most tumors can becharacterized as being defective in p53 function.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO. 1 is the human lrba siRNA (siRNA1), including 3′ two-dToverhang.

SEQ ID NO. 2 is the human lrba siRNA, including 3′ two-dT overhang.

SEQ ID NO. 3 is the murine LRBA-α amino acid sequence (FIG. 1A).

SEQ ID NO. 4 is the murine LRBA-β amino acid sequence (FIG. 1A).

SEQ ID NO. 5 is the additional amino acid sequence at end of LRBA-βprotein sequence (FIG. 1A).

SEQ ID NO. 6 is the murine LRBA-γ amino acid sequence (FIG. 1A).

SEQ ID NO. 7 is the additional amino acid sequence at end of LRBA-γprotein sequence (FIG. 1A).

SEQ ID NO. 8 is the human LRBA amino acid sequence also termed LRBA-α(FIGS. 9 and 3).

SEQ ID NO. 9 is the amino acid sequence of murine LRBA putative PKAbinding sites (FIG. 2A).

SEQ ID NO. 10 is the amino acid sequence of human LRBA putative PKAbinding sites (FIG. 2A).

SEQ ID NO. 11 is the amino acid sequence of drosophila LRBA putative PKAbinding sites (FIG. 2A).

SEQ ID NO. 12 is the amino acid sequence of drosophila LRBA2 putativePKA binding sites (FIG. 2A).

SEQ ID NO. 13 is the amino acid sequence of C. elegans LRBA putative PKAbinding sites (FIG. 2A).

SEQ ID NO. 14 is the C-terminal amino acid sequence of murine LRBA alsotermed LRBA-α (FIG. 3).

SEQ ID NO. 15 is the C-terminal amino acid sequence of human LRBA (FIG.3).

SEQ ID NO. 16 is the C-terminal amino acid sequence of drosophila LRBA(FIG. 3).

SEQ ID NO. 17 is the C-terminal amino acid sequence of human CHS1 (FIG.3).

SEQ ID NO. 18 is the C-terminal amino acid sequence of human FAN (FIG.3).

SEQ ID NO. 19 is the C-terminal amino acid sequence of murine LRBA-β(FIG. 3).

SEQ ID NO. 20 is the C-terminal amino acid sequence of murine LRBA-γ(FIG. 3).

SEQ ID NO. 21 is the human LRBA-β amino acid sequence (FIG. 9).

SEQ ID NO. 22 is the human LRBA-γ amino acid sequence (FIG. 9).

SEQ ID NO. 23 is the human LRBA-δ amino acid sequence (FIG. 9).

SEQ ID NO. 24 is the human LRBA-ε amino acid sequence (FIG. 9).

SEQ ID NO. 25 is the amino acid sequence of HSH domain of murine LRBA(FIG. 10).

SEQ ID NO. 26 is the amino acid sequence of HSH domain of human LRBA(FIG. 10).

SEQ ID NO. 27 is the amino acid sequence of HSH domain of drosophilaAKAP550 (FIG. 10).

SEQ ID NO. 28 is the amino acid sequence of HSH domain of C. elegansF10F2.1 (FIG. 10).

SEQ ID NO. 29 is the amino acid sequence of HSH domain of arabidopsisCDC4L (FIG. 10).

SEQ ID NO. 30 is the amino acid sequence of HSH domain of dictyosteliumLysA (FIG. 10).

SEQ ID NO. 31 is the amino acid sequence of HSH domain of arabidopsisLYSTL.

SEQ ID NO. 32 is the inserted amino acid sequence in human LRBA-γ.

SEQ ID NO. 33 is the 5′ end of human lrba-ε isoform with a long 5′ UTR(FIG. 15).

SEQ ID NO. 34 is the first 73 amino acids of the human LRBA (FIG. 15).

SEQ ID NO. 35 is the partial major coding sequence of human LRBA (FIG.15).

SEQ ID NO. 36 is the amino acids encoded by the extra exon interruptingthe lrba gene (FIG. 15).

SEQ ID NO. 37 is the promoter and part of the 5′ cDNA sequence of thehuman lrba gene (FIG. 17).

SEQ ID NO. 38 is the human lrba siRNA antisense strand, including 3′two-dT overhang.

SEQ ID NO. 39 is the human lrba siRNA (siRNA2), including 3′ two-dToverhang.

SEQ ID NOs. 40-46 are the primers used in cloning and sequencing ofmurine lrba cDNA.

SEQ ID NOs. 47-50 are the primers used in cloning and sequencing ofhuman lrba cDNA.

SEQ ID NOs. 51-56 are the primers used in RT-PCR analysis of murine lrbaexpression.

SEQ ID NOs. 48, 57-61 are the primers used for amplification of humanlrba.

SEQ ID NOs. 62-118 are the human lrba 5′ splice donor sites (exons 1-57)(Table 2).

SEQ ID NOs. 119-175 are the human lrba 3′ splice acceptor sites (introns1-57) (Table 2).

SEQ ID NO. 176 is the amino acid sequence of p21 RAS motif.

SEQ ID NO. 177 is the human lrba siRNA (siRNA1).

SEQ ID NO. 178 is the human lrba siRNA.

SEQ ID NO. 179 is the human lrba siRNA antisense strand.

SEQ ID NO. 180 is the human lrba siRNA (siRNA2).

SEQ ID NO:181 is the RNA sequence between the two open reading frames ofhuman lrbaδ (FIG. 16).

SEQ ID NO:182 is the predicted full-length amino acid sequence of humanLRBA (all five isoforms) (FIG. 9).

DETAILED DISCLOSURE OF THE INVENTION

The subject invention concerns a method of inhibiting cancerous tumorgrowth in a patient by suppressing lrba function. Preferably, the methodcomprises suppressing the functional expression of the lrba gene.Various methods known in the art for suppressing the functionalexpression of a gene can be utilized to carry out this method of thesubject invention. The lrba gene can be disrupted partially (e.g., aleaky mutation), resulting, for example, in reduced expression, or thelrba gene can be fully disrupted (e.g., complete gene ablation). Suchmutations can include, for example, point mutations, such as transitionsor transversions, or insertions and/or deletions, and the mutation canoccur in the coding region encoding lrba or merely in its regulatorysequences. According to the method of the subject invention, functionalexpression of the lrba gene can be suppressed at any level. In anotheraspect, the subject invention includes methods of disrupting expressionof the lrba gene in vivo or in vitro.

Using the method of the subject invention, lrba function is suppressed,which causes inhibition of tumor growth. Preferably, the suppression oflrba function results in death of tumor cells. More preferably, lrbafunction is suppressed to an extent that normal (non-cancerous) cellsare not killed.

Various means for suppression of lrba function can be utilized accordingto the method of the subject invention. For example, suppression of lrbafunction can be carried by administration of an agent that directly orindirectly causes suppression of lrba function. Agents suitable for themethod of the subject invention include nucleic acids, such as a geneticconstruct or other genetic means for directing expression of anantagonist of lrba function. Nucleic acid molecules suitable for themethod of the invention include, for example, anti-sensepolynucleotides, or other polynucleotides that bind to lrba mRNA, forexample. Preferably, the nucleic acid molecules administered to thepatient are those disclosed herein. Other agents that can be utilized tocarry out suppression of lrba function include, for example,peptidomimetics, ribozymes, and RNA aptamers.

According to the method of the subject invention, polypeptides can beadministered to a patient in order to suppress lrba function and inhibittumor growth. Preferably, the polypeptides utilized are those disclosedherein. More preferably, the polypeptides comprise fragments of thefull-length lrba amino acid sequence (including fragments of full-lengthamino acid sequences of lrba orthologs). Most preferably, thepolypeptides comprise amino acid sequences corresponding to the BEACHdomain, WD domain, or BEACH and WD domains, of the lrba gene (includinglrba gene orthologs). Various means for delivering polypeptides to acell can be utilized to carry out the method of the subject invention.For example, protein transduction domains (PTDs) can be fused to thepolypeptide, producing a fusion polypeptide, in which the PTDs arecapable of transducing the polypeptide cargo across the plasma membrane(Wadia, J. S. and Dowdy, S. F., Curr. Opin. Biotechnol., 2002,13(1)52-56). Examples of PTDs include the Drosophila homeotictranscription protein antennapedia (Antp), the herpes simples virusstructural protein VP22, and the human immuno-deficiency virus 1 (HIV-1)transcriptional activator Tat protein.

According to the method of tumor inhibition of the subject invention,recombinant cells can be administered to a patient, wherein therecombinant cells have been genetically modified to express an lrba geneproduct, such as a portion of the amino acid sequences set forth in FIG.1 (SEQ ID NOs. 3-7) or FIG. 9 (SEQ ID NOs. 8 and 21-24), or variantsthereof.

The method of tumor inhibition of the subject invention can be used totreat patient suffering from cancer or as a cancer preventative. Themethod of tumor inhibition of the subject invention can be used to treatpatients suffering from a variety of cancers including, but not limited,to cancer of the breast, prostate, melanoma, chronic myelogenousleukemia, cervical cancer, adenocarcinoma, lymphoblastic leukemia,colorectal cancer, and lung carcinoma.

In another aspect, the subject invention provides isolated and/orpurified nucleotide sequences comprising: (i) a polynucleotide sequenceencoding the amino acid sequence set forth in FIG. 1 (SEQ ID NOs. 3-7)or FIG. 9 (SEQ ID NOs. 8 and 21-24), or a complement thereof; (ii) apolynucleotide sequence having at least about 20% to 99.99% identity tothe polynucleotide sequence of (i); (iii) a polynucleotide encoding afragment of the amino acid sequence shown in FIG. 1 (SEQ ID NOs 3-7) orFIG. 9 (SEQ ID NOs. 8 and 21-24); or (iv) an interfering RNA sequencecorresponding to the transcript of the polynucleotide set forth in FIG.1 (SEQ ID NOs. 3-7) or FIG. 9 (SEQ ID NOs. 8 and 21-24), or a fragmentof the transcript.

Nucleotide, polynucleotide, or nucleic acid sequences(s) are understoodto mean, according to the present invention, either a double-strandedDNA, a single-stranded DNA, or products of transcription of the saidDNAs (e.g., RNA molecules). It should also be understood that thepresent invention does not relate to the genomic nucleotide sequencesencoding lrba in their natural/native environment or natural/nativestate. The nucleic acid, polynucleotide, or nucleotide sequences of theinvention have been isolated, purified (or partially purified), byseparation methods including, but not limited to, ion-exchangechromatography, molecular size exclusion chromatography, affinitychromatography, or by genetic engineering methods such as amplification,cloning or subcloning.

Optionally, the polynucleotide sequences of the instant invention canalso contain one or more polynucleotides encoding heterologouspolypeptide sequences (e.g., tags that facilitate purification of thepolypeptides of the invention (see, for example, U.S. Pat. No.6,342,362, hereby incorporated by reference in its entirety; Altendorfet al. [1999-WWW, 2000] “Structure and Function of the F_(o) Complex ofthe ATP Synthase from Escherichia Coli,” J. of Experimental Biology203:19-28, The Co. of Biologists, Ltd., G.B.; Baneyx [1999] “RecombinantProtein Expression in Escherichia coli,” Biotechnology 10:411-21,Elsevier Science Ltd.; Eihauer et al. [2001] “The FLAG™ Peptide, aVersatile Fusion Tag for the Purification of Recombinant Proteins,” J.Biochem Biophys Methods 49:455-65; Jones et al. [1995] J. Chromatography707:3-22; Jones et al. [1995] “Current Trends in Molecular Recognitionand Bioseparation,” J. of Chromatography A. 707:3-22, Elsevier ScienceB.V.; Margolin [2000] “Green Fluorescent Protein as a Reporter forMacromolecular Localization in Bacterial Cells,” Methods 20:62-72,Academic Press; Puig et al. [2001] “The Tandem Affinity Purification(TAP) Method: A General Procedure of Protein Complex Purification,”Methods 24:218-29, Academic Press; Sassenfeld [1990] “EngineeringProteins for Purification,” TibTech 8:88-93; Sheibani [1999]“Prokaryotic Gene Fusion Expression Systems and Their Use in Structuraland Functional Studies of Proteins,” Prep. Biochem. & Biotechnol.29(1):77-90, Marcel Dekker, Inc.; Skerra et al. [1999] “Applications ofa Peptide Ligand for Streptavidin: the Strep-tag”, BiomolecularEngineering 16:79-86, Elsevier Science, B.V.; Smith [1998] “Cookbook forEukaryotic Protein Expression: Yeast, Insect, and Plant ExpressionSystems,” The Scientist 12(22):20; Smyth et al. [2000] “EukaryoticExpression and Purification of Recombinant Extracellular Matrix ProteinsCarrying the Strep II Tag”, Methods in Molecular Biology, 139:49-57;Unger [1997] “Show Me the Money: Prokaryotic Expression Vectors andPurification Systems,” The Scientist 11(17):20, each of which is herebyincorporated by reference in their entireties), or commerciallyavailable tags from vendors such as such as STRATAGENE (La Jolla,Calif.), NOVAGEN (Madison, Wis.), QIAGEN, Inc., (Valencia, Calif.), orINVITROGEN (San Diego, Calif.).

Other aspects of the invention provide vectors containing one or more ofthe polynucleotides of the invention. The vectors can be vaccine,replication, or amplification vectors. In some embodiments of thisaspect of the invention, the polynucleotides are operably associatedwith regulatory elements capable of causing the expression of thepolynucleotide sequences. Such vectors include, among others,chromosomal, episomal and virus-derived vectors, e.g., vectors derivedfrom bacterial plasmids, from bacteriophage, from transposons, fromyeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations of theaforementioned vector sources, such as those derived from plasmid andbacteriophage genetic elements (e.g., cosmids and phagemids).

As indicated above, vectors of this invention can also comprise elementsnecessary to provide for the expression and/or the secretion of apolypeptide encoded by the nucleotide sequences of the invention in agiven host cell. The vector can contain one or more elements selectedfrom the group consisting of a promoter, signals for initiation oftranslation, signals for termination of translation, and appropriateregions for regulation of transcription. In certain embodiments, thevectors can be stably maintained in the host cell and can, optionally,contain signal sequences directing the secretion of translated protein.Other embodiments provide vectors that are not stable in transformedhost cells. Vectors can integrate into the host genome or beautonomously-replicating vectors.

In a specific embodiment, a vector comprises a promoter operably linkedto a protein or peptide-encoding nucleic acid sequence, one or moreorigins of replication, and, optionally, one or more selectable markers(e.g., an antibiotic resistance gene). Non-limiting exemplary vectorsfor the expression of the polypeptides of the invention include pBr-typevectors, pET-type plasmid vectors (Promega), pBAD plasmid vectors(Invitrogen) or those provided in the examples below. Furthermore,vectors according to the invention are useful for transforming hostcells for the cloning or expression of the nucleotide sequences of theinvention.

Promoters which may be used to control expression include, but are notlimited to, the CMV promoter, the SV40 early promoter region (Bemoistand Chambon [1981] Nature 290:304-310), the promoter contained in the 3′long terminal repeat of Rous sarcoma virus (Yamamoto, et al. [1980] Cell22:787-797), the herpes thymidine kinase promoter (Wagner et al. [1981]Proc. Natl. Acad. Sci. USA 78:1441-1445), the regulatory sequences ofthe metallothionein gene (Brinster et al. [1982] Nature 296:39-42);prokaryotic vectors containing promoters such as the β-lactamasepromoter (Villa-Kamaroff, et al. [1978] Proc. Natl. Acad. Sci. USA75:3727-3731), or the tac promoter (DeBoer, et al. [1983] Proc. Natl.Acad. Sci. USA 80:21-25); see also, “Useful Proteins from RecombinantBacteria” in Scientific American, 1980, 242:74-94; plant expressionvectors comprising the nopaline synthetase promoter region(Herrera-Estrella et al. [1983] Nature 303:209-213) or the cauliflowermosaic virus 35S RNA promoter (Gardner, et al. [1981] Nucl. Acids Res.9:2871), and the promoter of the photosynthetic enzyme ribulosebiphosphate carboxylase (Herrera-Estrella et al. [1984] Nature310:115-120); promoter elements from yeast or fungi such as the Gal 4promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerolkinase) promoter, and/or the alkaline phosphatase promoter.

The subject invention also provides for “homologous” or “modified”nucleotide sequences. Modified nucleic acid sequences will be understoodto mean any nucleotide sequence obtained by mutagenesis according totechniques well known to persons skilled in the art, and exhibitingmodifications in relation to the normal sequences. For example,mutations in the regulatory and/or promoter sequences for the expressionof a polypeptide that result in a modification of the level ofexpression of a polypeptide according to the invention provide for a“modified nucleotide sequence”. Likewise, substitutions, deletions, oradditions of nucleic acid to the polynucleotides of the inventionprovide for “homologous” or “modified” nucleotide sequences. In variousembodiments, “homologous” or “modified” nucleic acid sequences havesubstantially the same biological or serological activity as the native(naturally occurring) LRBA polypeptides. A “homologous” or “modified”nucleotide sequence will also be understood to mean a splice variant ofthe polynucleotides of the instant invention or any nucleotide sequenceencoding a “modified polypeptide” as defined below.

A homologous nucleotide sequence, for the purposes of the presentinvention, encompasses a nucleotide sequence having a percentageidentity with the bases of the nucleotide sequences of between at least(or at least about) 20.00% to 99.99% (inclusive). The aforementionedrange of percent identity is to be taken as including, and providingwritten description and support for, any fractional percentage, inintervals of 0.01%, between 20.00% and 99.99%. These percentages arepurely statistical and differences between two nucleic acid sequencescan be distributed randomly and over the entire sequence length.

In various embodiments, homologous sequences exhibiting a percentageidentity with the bases of the nucleotide sequences of the presentinvention can have 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identitywith the polynucleotide sequences of the instant invention.

Both protein and nucleic acid sequence homologies may be evaluated usingany of the variety of sequence comparison algorithms and programs knownin the art. Such algorithms and programs include, but are by no meanslimited to, TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (Pearson andLipman [1988] Proc. Natl. Acad. Sci. USA 85(8):2444-2448; Altschul etal. [1990] J. Mol. Biol. 215(3):403-410; Thompson et al. [1994] NucleicAcids Res. 22(2):4673-4680; Higgins et al. [1996] Methods Enzymol.266:383-402; Altschul et al. [1990] J. Mol. Biol. 215(3):403-410;Altschul et al. [1993] Nature Genetics 3:266-272).

The subject invention also provides nucleotide sequences complementaryto any of the polynucleotide sequences disclosed herein. Thus, theinvention is understood to include any DNA whose nucleotides arecomplementary to those of the sequence of the invention, and whoseorientation is reversed (e.g., an antisense sequence).

The present invention further provides fragments of the polynucleotidesequences provided herein. Representative fragments of thepolynucleotide sequences according to the invention will be understoodto mean any nucleotide fragment having at least 8 or 9 successivenucleotides, preferably at least 12 successive nucleotides, and stillmore preferably at least 15 or at least 20 successive nucleotides of thesequence from which it is derived. The upper limit for such fragments isthe total number of polynucleotides found in the full-length sequence(or, in certain embodiments, of the full length open reading frame (ORF)identified herein). It is understood that such fragments refer only toportions of the disclosed polynucleotide sequences that are not listedin a publicly available database or prior art references. However, itshould be understood that with respect to the method for inhibitingtumor growth of the subject invention, disclosed nucleotides (andpolypeptides encoded by such nucleotides) that are listed in a publiclyavailable database or prior art reference can also be utilized. Forexample, nucleotide sequences that are lrba orthologs, or fragmentsthereof, which have been previously identified, can be utilized to carryout the method for inhibiting tumor growth of the subject invention.Thus sequences from the drosophila melanogaster genomic sequence(GENBANK accession number AE003433), cLRBA (GENBANK accession numberT20719, Caenorhabditis elegans), aCDC4L (GENBANK accession numberT00867, Arabidopsis thaliana), LSVA (GENBANK accession number AAD52096,Dictyostelium discoideum), hFAN (GENBANK accession number NP_(—)0035711,Homo sapiens), CHS1 (Chediak-Higashi Syndrome 1, GENBANK accessionnumber NP_(—)000072, Homo sapiens), or mBG (GENBANK accession numberAAB60778, Mus musculus) can be utilized to carry out the method of tumorgrowth inhibition of the subject invention.

In other embodiments, fragments contain from one nucleotide less thanthe full length polynucleotide sequence (1249 nucleotides) to fragmentscomprising up to, and including 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150,155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220,225, 230, 235, 240, 245, 250, 255, . . . and up to, for example, 1,245consecutive nucleotides of a particular sequence disclosed herein.

Yet other embodiments provide fragments (or detection probes) comprisingnucleotides within the lrba cDNA sequence, such as the human lrba cDNAsequence (GenBank accession number NM_(—)006726), including 245 to 458(G-peptide), 488 to 1424 (HSH domain), 2573-2627 (siRNA1) (SEQ ID NO.5), 3179 to 4148 (SET domain), 4301 to 4505 (PKA RII binding sites),6347 to 6749 (WDL repeats), 6878 to 7709 (BEACH domain), 8018 to 8831(WD repeats).

Among these representative fragments, those capable of hybridizing understringent conditions with a nucleotide sequence according to theinvention are preferred. Conditions of high or intermediate stringencyare provided infra and are chosen to allow for hybridization between twocomplementary DNA fragments. Hybridization conditions for apolynucleotide of about 300 bases in size will be adapted by personsskilled in the art for larger- or smaller-sized oligonucleotides,according to methods well known in the art (see, for example, Sambrooket al. [ 1989]).

The subject invention also provides detection probes (e.g., fragments ofthe disclosed polynucleotide sequences) for hybridization with a targetsequence or an amplicon generated from the target sequence. Such adetection probe will advantageously have as sequence a sequence of atleast 9, 12, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, or 100 nucleotides. Alternatively, detection probes can comprise8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110,115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180,185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250,255, . . . and up to, for example, 1245 consecutive nucleotides of thedisclosed nucleic acids. The detection probes can also be used aslabeled probe or primer in the subject invention. Labeled probes orprimers are labeled with a radioactive compound or with another type oflabel. Alternatively, non-labeled nucleotide sequences may be useddirectly as probes or primers; however, the sequences are generallylabeled with a radioactive element (³²P, ³⁵S, ³H, ¹²⁵I) or with amolecule such as biotin, acetylaminofluorene, digoxigenin,5-bromodeoxyuridine, or fluorescein to provide probes that can be usedin numerous applications.

The nucleotide sequences according to the invention may also be used inanalytical systems, such as DNA chips. DNA chips and their uses are wellknown in the art and (see for example, U.S. Pat. Nos. 5,561,071;5,753,439; 6,214,545; Schena et al. [1996] BioEssays 18:427-431; Bianchiet al. [1997] Clin. Diagn. Virol. 8:199-208; each of which is herebyincorporated by reference in their entireties) and/or are provided bycommercial vendors such as AFFYMETRIX, Inc. (Santa Clara, Calif.).

Various degrees of stringency of hybridization can be employed. The moresevere the conditions, the greater the complementarity that is requiredfor duplex formation. Severity of conditions can be controlled bytemperature, probe concentration, probe length, ionic strength, time,and the like. Preferably, hybridization is conducted under moderate tohigh stringency conditions by techniques well known in the art, asdescribed, for example, in Keller, G. H., M. M. Manak [1987] DNA Probes,Stockton Press, New York, N.Y., pp. 169-170.

By way of example, hybridization of immobilized DNA on Southern blotswith ³²P-labeled gene-specific probes can be performed by standardmethods (Maniatis et al. [1982] Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory, New York). In general, hybridization andsubsequent washes can be carried out under moderate to high stringencyconditions that allow for detection of target sequences with homology tothe exemplified polynucleotide sequence. For double-stranded DNA geneprobes, hybridization can be carried out overnight at 20-25° C. belowthe melting temperature (Tm) of the DNA hybrid in 6× SSPE, 5× Denhardt'ssolution, 0.1% SDS, 0.1 mg/ml denatured DNA. The melting temperature isdescribed by the following formula (Beltz et al. [1983] Methods ofEnzymology, R. Wu, L. Grossman and K. Moldave [eds.] Academic Press, NewYork 100:266-285).T _(m)=81.5° C.+16.6 Log[Na+]+0.41(% G+C)−0.61(% formamide)−600/lengthof duplex in base pairs.

Washes are typically carried out as follows:

-   -   (1) twice at room temperature for 15 minutes in 1× SSPE, 0.1%        SDS (low stringency wash);    -   (2) once at T_(m)-20° C. for 15 minutes in 0.2× SSPE, 0.1% SDS        (moderate stringency wash).

For oligonucleotide probes, hybridization can be carried out overnightat 10-20° C. below the melting temperature (T_(m)) of the hybrid in 6×SSPE, 5× Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA. T_(m)for oligonucleotide probes can be determined by the following formula:

T_(m) (° C.)=2(number T/A base pairs)+4(number G/C base pairs) (Suggs etal. [1981] ICN-UCLA Symp. Dev. Biol. Using Purified Genes, D. D. Brown[ed.], Academic Press, New York, 23:683-693).

Washes can be carried out as follows:

-   -   (1) twice at room temperature for 15 minutes 1× SSPE, 0.1% SDS        (low stringency wash;    -   2) once at the hybridization temperature for 15 minutes in 1×        SSPE, 0.1% SDS (moderate stringency wash).

In general, salt and/or temperature can be altered to change stringency.With a labeled DNA fragment >70 or so bases in length, the followingconditions can be used:

Low: 1 or 2X SSPE, room temperature Low: 1 or 2X SSPE, 42° C. Moderate:0.2X or 1X SSPE, 65° C. High: 0.1X SSPE, 65° C.

By way of another non-limiting example, procedures using conditions ofhigh stringency can also be performed as follows: Pre-hybridization offilters containing DNA is carried out for 8 h to overnight at 65° C. inbuffer composed of 6× SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02%PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/ml denatured salmon sperm DNA.Filters are hybridized for 48 h at 65° C., the preferred hybridizationtemperature, in pre-hybridization mixture containing 100 μg/ml denaturedsalmon sperm DNA and 5-20×10⁶ cpm of ³²P-labeled probe. Alternatively,the hybridization step can be performed at 65° C. in the presence of SSCbuffer, 1× SSC corresponding to 0.15M NaCl and 0.05 M Na citrate.Subsequently, filter washes can be done at 37° C. for 1 h in a solutioncontaining 2× SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA, followed by awash in 0.1× SSC at 50° C. for 45 min. Alternatively, filter washes canbe performed in a solution containing 2× SSC and 0.1% SDS, or 0.5× SSCand 0.1% SDS, or 0.1× SSC and 0.1% SDS at 68° C. for 15 minuteintervals. Following the wash steps, the hybridized probes aredetectable by autoradiography. Other conditions of high stringency whichmay be used are well known in the art (see, for example, Sambrook et al.[1989] Molecular Cloning, A Laboratory Manual, Second Edition, ColdSpring Harbor Press, N.Y., pp. 9.47-9.57; and Ausubel et al. [1989]Current Protocols in Molecular Biology, Green Publishing Associates andWiley Interscience, N.Y., each incorporated herein in its entirety).

A further non-limiting example of procedures using conditions ofintermediate stringency are as follows: Filters containing DNA arepre-hybridized, and then hybridized at a temperature of 60° C. in thepresence of a 5× SSC buffer and labeled probe. Subsequently, filterswashes are performed in a solution containing 2× SSC at 50° C. and thehybridized probes are detectable by autoradiography. Other conditions ofintermediate stringency which may be used are well known in the art(see, for example, Sambrook et al. [1989] Molecular Cloning, ALaboratory Manual, Second Edition, Cold Spring Harbor Press, N.Y., pp.9.47-9.57; and Ausubel et al. [1989] Current Protocols in MolecularBiology, Green Publishing Associates and Wiley Interscience, N.Y., eachof which is incorporated herein in its entirety).

Duplex formation and stability depend on substantial complementaritybetween the two strands of a hybrid and, as noted above, a certaindegree of mismatch can be tolerated. Therefore, the probe sequences ofthe subject invention include mutations (both single and multiple),deletions, insertions of the described sequences, and combinationsthereof, wherein said mutations, insertions and deletions permitformation of stable hybrids with the target polynucleotide of interest.Mutations, insertions and deletions can be produced in a givenpolynucleotide sequence in many ways, and these methods are known to anordinarily skilled artisan. Other methods may become known in thefuture.

It is also well known in the art that restriction enzymes can be used toobtain functional fragments of the subject DNA sequences. For example,Bal31 exonuclease can be conveniently used for time-controlled limiteddigestion of DNA (commonly referred to as “erase-a-base” procedures).See, for example, Maniatis et al. [1982] Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory, New York; Wei et al. [1983] J.Biol. Chem. 258:13006-13512. The nucleic acid sequences of the subjectinvention can also be used as molecular weight markers in nucleic acidanalysis procedures.

The invention also provides host cells transformed by a polynucleotideaccording to the invention and the production of LRBA (or LRBA ortholog)polypeptides by the transformed host cells. In some embodiments,transformed cells comprise an expression vector containing LRBA, or LRBAortholog, polynucleotide sequences. Other embodiments provide for hostcells transformed with nucleic acids. Yet other embodiments providetransformed cells comprising an expression vector containing fragmentsof lrba, or lrba ortholog, polynucleotide sequences. Transformed hostcells according to the invention are cultured under conditions allowingthe replication and/or the expression of the nucleotide sequences of theinvention. Expressed polypeptides are recovered from culture media andpurified, for further use, according to methods known in the art.

The host cell may be chosen from eukaryotic or prokaryotic systems, forexample bacterial cells (Gram negative or Gram positive), yeast cells,animal cells, plant cells, and/or insect cells using baculovirusvectors. In some embodiments, the host cell for expression of thepolypeptides include, and are not limited to, those taught in U.S. Pat.Nos. 6,319,691; 6,277,375; 5,643,570; 5,565,335; Unger [1997] TheScientist 11(17):20; or Smith [1998] The Scientist 12(22):20, each ofwhich is incorporated by reference in its entirety, including allreferences cited within each respective patent or reference. Otherexemplary, and non-limiting, host cells include Staphylococcus spp.,Enterococcus spp., E. coli, and Bacillus subtilis; fungal cells, such asStreptomyces spp., Aspergillus spp., S. cerevisiae, Schizosaccharomycespombe, Pichia pastoris, Hansela polymorpha, Kluveromyces lactis, andYarrowia lipolytica; insect cells such as Drosophila S2 and SpodopteraSf9 cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 andBowes melanoma cells; and plant cells. A great variety of expressionsystems can be used to produce the polypeptides of the invention andpolynucleotides can be modified according to methods known in the art toprovide optimal codon usage for expression in a particular expressionsystem.

Furthermore, a host cell strain may be chosen that modulates theexpression of the inserted sequences, modifies the gene product, and/orprocesses the gene product in the specific fashion. Expression fromcertain promoters can be elevated in the presence of certain inducers;thus, expression of the genetically engineered polypeptide may becontrolled. Furthermore, different host cells have characteristic andspecific mechanisms for the translational and post-translationalprocessing and modification (e.g., glycosylation, phosphorylation) ofproteins. Appropriate cell lines or host systems can be chosen to ensurethe desired modification and processing of the foreign proteinexpressed. For example, expression in a bacterial system can be used toproduce an unglycosylated core protein product whereas expression inyeast will produce a glycosylated product. Expression in mammalian cellscan be used to provide “native” glycosylation of a heterologous protein.Furthermore, different vector/host expression systems may effectprocessing reactions to different extents.

Nucleic acids and/or vectors can be introduced into host cells bywell-known methods, such as, calcium phosphate transfection,DEAE-dextran mediated transfection, transfection, microinjection,cationic lipid-mediated transfection, electroporation, transduction,scrape loading, ballistic introduction and infection (see, for example,Sambrook et al. [1989] Molecular Cloning: A Laboratory Manual, 2^(nd)Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

The subject invention also provides for the expression of a polypeptide,derivative, or a variant (e.g., a splice variant) encoded by apolynucleotide sequence disclosed herein. Alternatively, the inventionprovides for the expression of a polypeptide fragment obtained from apolypeptide, derivative, or a variant encoded by a polynucleotidefragment derived from the polynucleotide sequences disclosed herein. Ineither embodiment, the disclosed sequences can be regulated by a secondnucleic acid sequence so that the polypeptide or fragment is expressedin a host transformed with a recombinant DNA molecule according to thesubject invention. For example, expression of a protein or peptide maybe controlled by any promoter/enhancer element known in the art.

The subject invention also provides nucleic acid based methods for theidentification of the presence of the lrba gene, or orthologs thereof,in a sample. These methods can utilize the nucleic acids of the subjectinvention and are well known to those skilled in the art (see, forexample, Sambrook et al. [1989] or Abbaszadega [2001] “AdvancedDetection of Viruses and Protozoan Parasites in Water,” Reviews inBiology and Biotechnology, 1(2):21-26). Among the techniques useful insuch methods are enzymatic gene amplification (or PCR), Southern blots,Northern blots, or other techniques utilizing nucleic acid hybridizationfor the identification of polynucleotide sequences in a sample. Thenucleic acids can be used to screen individuals for cancers, tumors, ormalignancies associated with dysregulation of the lrba gene or itstranscriptional products.

The subject invention also provides polypeptides encoded by nucleotidesequences of the invention. The subject invention also providesfragments of at least 5 amino acids of a polypeptide encoded by thepolynucleotides of the instant invention.

In the context of the instant invention, the terms polypeptide, peptideand protein are used interchangeably. Likewise, the terms variant andhomologous are also used interchangeably. It should be understood thatthe invention does not relate to the polypeptides in natural form ornative environment. Peptides and polypeptides according to the inventionhave been isolated or obtained by purification from natural sources (ortheir native environment), chemically synthesized, or obtained from hostcells prepared by genetic manipulation (e.g., the polypeptides, orfragments thereof, are recombinantly produced by host cells).Polypeptides according to the instant invention may also containnon-natural amino acids, as will be described below.

“Variant” or “homologous” polypeptides will be understood to designatethe polypeptides containing, in relation to the native polypeptide,modifications such as deletion, addition, or substitution of at leastone amino acid, truncation, extension, or the addition of chimericheterologous polypeptides. Optionally, “variant” or “homologous”polypeptides can contain a mutation or post-translational modifications.Among the “variant” or “homologous” polypeptides, those whose amino acidsequence exhibits 20.00% to 99.99% (inclusive) identity to the nativepolypeptide sequence are preferred. The aforementioned range of percentidentity is to be taken as including, and providing written descriptionand support for, any fractional percentage, in intervals of 0.01%,between 50.00% and, up to, including 99.99%. These percentages arepurely statistical and differences between two polypeptide sequences canbe distributed randomly and over the entire sequence length.

“Variant” or “homologous” polypeptide sequences exhibiting a percentageidentity with the polypeptides of the present invention can,alternatively, have 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 91, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identitywith the polypeptide sequences of the instant invention. The expressionequivalent amino acid is intended here to designate any amino acidcapable of being substituted for one of the amino acids in the basicstructure without, however, essentially modifying the biologicalactivities of the corresponding peptides and as provided below.

By way of example, amino acid substitutions can be carried out withoutresulting in a substantial modification of the biological activity ofthe corresponding modified polypeptides; for example, the replacement ofleucine with valine or isoleucine; aspartic acid with glutamic acid;glutamine with asparagine; arginine with lysine; and the reversesubstitutions can be performed without substantial modification of thebiological activity of the polypeptides.

In other embodiments, homologous polypeptides according to the subjectinvention also include various splice variants identified within thelrba coding sequence.

The subject invention also provides biologically active fragments of apolypeptide according to the invention and includes those peptidescapable of eliciting an immune response. The immune response can providecomponents (either antibodies or components of the cellular immuneresponse (e.g., B-cells, helper, cytotoxic, and/or suppressor T-cells)reactive with the biologically active fragment of a polypeptide, theintact, full length, unmodified polypeptide disclosed herein, or boththe biologically active fragment of a polypeptide and the intact, fulllength, unmodified polypeptides disclosed herein. Biologically activefragments according to the invention comprise from five (5) amino acidsto one amino acid less than the full length of any polypeptide sequenceprovided herein. Alternatively, fragments comprising 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120,125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190,195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, . . .and up to 2845 consecutive amino acids of a disclosed polypeptidesequence are provided herein.

Fragments, as described herein, can be obtained by cleaving thepolypeptides of the invention with a proteolytic enzyme (such astrypsin, chymotrypsin, or collagenase) or with a chemical reagent, suchas cyanogen bromide (CNBr). Alternatively, polypeptide fragments can begenerated in a highly acidic environment, for example at pH 2.5. Suchpolypeptide fragments may be equally well prepared by chemical synthesisor using hosts transformed with an expression vector containing nucleicacids encoding polypeptide fragments according to the invention. Thetransformed host cells contain a nucleic acid and are cultured accordingto well-known methods; thus, the invention allows for the expression ofthese fragments, under the control of appropriate elements forregulation and/or expression of the polypeptide fragments.

Modified polypeptides according to the invention are understood todesignate a polypeptide obtained by variation in the splicing oftranscriptional products of the lrba gene, genetic recombination, or bychemical synthesis as described below. Modified polypeptides contain atleast one modification in relation to the normal polypeptide sequence.These modifications can include the addition, substitution, deletion ofamino acids contained within the polypeptides of the invention.

Conservative substitutions whereby an amino acid of one class isreplaced with another amino acid of the same type fall within the scopeof the subject invention so long as the substitution does not materiallyalter the biological activity of the polypeptide. For example, the classof nonpolar amino acids include Ala, Val, Leu, Ile, Pro, Met, Phe, andTrp; the class of uncharged polar amino acids includes Gly, Ser, Thr,Cys, Tyr, Asn, and Gln; the class of acidic amino acids includes Asp andGlu; and the class of basic amino acids includes Lys, Arg, and His. Insome instances, non-conservative substitutions can be made where thesesubstitutions do not significantly detract from the biological activityof the polypeptide.

In order to extend the life of the polypeptides of the invention, it maybe advantageous to use non-natural amino acids, for example in the Dform, or alternatively amino acid analogs, such as sulfur-containingforms of amino acids. Alternative means for increasing the life ofpolypeptides can also be used in the practice of the instant invention.For example, polypeptides of the invention, and fragments thereof, canbe recombinantly modified to include elements that increase the plasma,or serum half-life of the polypeptides of the invention. These elementsinclude, and are not limited to, antibody constant regions (see forexample, U.S. Pat. No. 5,565,335, hereby incorporated by reference inits entirety, including all references cited therein), or other elementssuch as those disclosed in U.S. Pat. Nos. 6,319,691; 6,277,375; or5,643,570, each of which is incorporated by reference in its entirety,including all references cited within each respective patent.Alternatively, the polynucleotides and genes of the instant inventioncan be recombinantly fused to elements that are useful in thepreparation of immunogenic constructs for the purposes of vaccineformulation or elements useful for the isolation of the polypeptides ofthe invention.

The polypeptides, fragments, and immunogenic fragments of the inventionmay further contain linkers that facilitate the attachment of thefragments to a carrier molecule for the stimulation of an immuneresponse or diagnostic purposes. The linkers can also be used to attachfragments according to the invention to solid support matrices for usein affinity purification protocols. In this aspect of the invention, thelinkers specifically exclude, and are not to be considered anticipated,where the fragment is a subsequence of another peptide, polypeptide, orprotein as identified in a search of protein sequence databases asindicated in the preceding paragraph. In other words, the non-identicalportions of the other peptide, polypeptide, of protein is not consideredto be a “linker” in this aspect of the invention. Non-limiting examplesof “linkers” suitable for the practice of the invention include chemicallinkers (such as those sold by Pierce, Rockford, Ill.), peptides thatallow for the connection of the immunogenic fragment to a carriermolecule (see, for example, linkers disclosed in U.S. Pat. Nos.6,121,424; 5,843,464; 5,750,352; and 5,990,275, hereby incorporated byreference in their entirety). In various embodiments, the linkers can beup to 50 amino acids in length, up to 40 amino acids in length, up to 30amino acids in length, up to 20 amino acids in length, up to 10 aminoacids in length, or up to 5 amino acids in length.

In other specific embodiments, the polypeptides, peptides, derivatives,or analogs thereof may be expressed as a fusion, or chimeric proteinproduct (comprising the protein, fragment, analog, or derivative joinedvia a peptide bond to a heterologous protein sequence (e.g., a differentprotein)). Such a chimeric product can be made by ligating theappropriate nucleic acid sequences encoding the desired amino acidsequences to each other by methods known in the art, in the propercoding frame, and expressing the chimeric product by methods commonlyknown in the art (see, for example, U.S. Pat. No. 6,342,362, herebyincorporated by reference in its entirety; Altendorf et al. [1999-WWW,2000] “Structure and Function of the F_(o) Complex of the ATP Synthasefrom Escherichia Coli,” J. of Experimental Biology 203:19-28, The Co. ofBiologists, Ltd., G.B.; Baneyx [1999] “Recombinant Protein Expression inEscherichia coli,” Biotechnology 10:411-21, Elsevier Science Ltd.;Eihauer et al. [2001] “The FLAG™ Peptide, a Versatile Fusion Tag for thePurification of Recombinant Proteins,” J. Biochem Biophys Methods49:455-65; Jones et al. [1995] J. Chromatography 707:3-22; Jones et al.[1995] “Current Trends in Molecular Recognition and Bioseparation,” J.Chromatography A. 707:3-22, Elsevier Science B.V.; Margolin [2000]“Green Fluorescent Protein as a Reporter for Macromolecular Localizationin Bacterial Cells,” Methods 20:62-72, Academic Press; Puig et al.[2001] “The Tandem Affinity Purification (TAP) Method: A GeneralProcedure of Protein Complex Purification,” Methods 24:218-29, AcademicPress; Sassenfeld [1990] “Engineering Proteins for Purification,”TibTech 8:88-93; Sheibani [1999] “Prokaryotic Gene Fusion ExpressionSystems and Their Use in Structural and Functional Studies of Proteins,”Prep. Biochem. & Biotechnol. 29(1):77-90, Marcel Dekker, Inc.; Skerra etal. [1999] “Applications of a Peptide Ligand for Streptavidin: TheStrep-tag”, Biomolecular Engineering 16:79-86, Elsevier Science, B.V.;Smith [1998] “Cookbook for Eukaryotic Protein Expression: Yeast, Insect,and Plant Expression Systems,” The Scientist 12(22):20; Smyth et al.[2000] “Eukaryotic Expression and Purification of RecombinantExtracellular Matrix Proteins Carrying the Strep II Tag”, Methods inMolecular Biology, 139:49-57; Unger [1997] “Show Me the Money:Prokaryotic Expression Vectors and Purification Systems,” The Scientist11(17):20, each of which is hereby incorporated by reference in theirentireties). Alternatively, such a chimeric product may be made byprotein synthetic techniques, e.g., by use of a peptide synthesizer.Fusion peptides can comprise polypeptides of the subject invention andone or more protein transduction domains, as described above. Suchfusion peptides are particularly useful for delivering the cargopolypeptide through the cell membrane.

The expression of the lrba gene or lrba gene product (e.g., DNA, RNA, orpolypeptide) is disregulated in a variety of cancers, tumors, and/ormalignancies. Non-limiting examples of such cancers, tumors, and/ormalignancies include prostate cancer, breast cancer, melanoma, chronicmyelogenous leukemia, cervical cancer, adenocarcinomas, lymphoblasticleukemia, colorectal cancer, and lung carcinoma. Accordingly, thepresent invention provides a method for screening, or aiding in thediagnosis of, an individual suspected of having a malignancy or cancer.The subject invention provides methods comprising the steps ofdetermining the amount of lrba in a biological sample obtained from saidindividual and comparing the measured amount of lrba to the amount oflrba found in the normal population. The presence of a significantlyincreased amount of lrba is associated with an indication of amalignancy or cancer. Lrba gene product can be detected by well-knownmethodologies including, and not limited to, Western blots, enzymelinked immunoassays (ELISAs), radioimmunoassays (RIAs), Northern blots,Southern blots, PCR-based assays, or other assays for the quantificationof gene product known to the skilled artisan. This information, inconjunction with other information available to the skilledpractitioner, assists in making a diagnosis.

Antisense technology can also be used to interfere with expression ofthe disclosed polynucleotides. For example, the transformation of a cellor organism with the reverse complement of a gene encoded by apolynucleotide exemplified herein can result in strand co-suppressionand silencing or inhibition of a target gene, e.g., one involved in theinfection process.

Polynucleotides disclosed herein are useful as target genes for thesynthesis of antisense RNA or dsRNA useful for RNA-mediated geneinterference. The ability to specifically inhibit gene function in avariety of organisms utilizing antisense RNA or ds RNA-mediatedinterference is well known in the fields of molecular biology (see forexample C. P. Hunter, Current Biology [1999] 9:R440-442; Hamilton etal., [1999] Science, 286:950-952; and S. W. Ding, Current Opinions inBiotechnology [2000] 11:152-156, hereby incorporated by reference intheir entireties). dsRNA (RNAi) typically comprises a polynucleotidesequence identical or homologous to a target gene (or fragment thereof)linked directly, or indirectly, to a polynucleotide sequencecomplementary to the sequence of the target gene (or fragment thereof).The dsRNA may comprise a polynucleotide linker sequence of sufficientlength to allow for the two polynucleotide sequences to fold over andhybridize to each other; however, a linker sequence is not necessary.The linker sequence is designed to separate the antisense and sensestrands of RNAi significantly enough to limit the effects of sterichindrances and allow for the formation of dsRNA molecules and should nothybridize with sequences within the hybridizing portions of the dsRNAmolecule. The specificity of this gene silencing mechanism appears to beextremely high, blocking expression only of targeted genes, whileleaving other genes unaffected. Accordingly, one method for controllinggene expression according to the subject invention provides materialsand methods using double-stranded interfering RNA (dsRNAi), orRNA-mediated interference (RNAi). The terms “dsRNAi”, “RNAi”, “iRNA”,and “siRNA” are used interchangeably herein unless otherwise noted.

RNA containing a nucleotide sequence identical to a fragment of thetarget gene is preferred for inhibition; however, RNA sequences withinsertions, deletions, and point mutations relative to the targetsequence can also be used for inhibition. Sequence identity mayoptimized by sequence comparison and alignment algorithms known in theart (see Gribskov and Devereux, Sequence Analysis Primer, StocktonPress, 1991, and references cited therein) and calculating the percentdifference between the nucleotide sequences by, for example, theSmith-Waterman algorithm as implemented in the BESTFIT software programusing default parameters (e.g., University of Wisconsin GeneticComputing Group). Alternatively, the duplex region of the RNA may bedefined functionally as a nucleotide sequence that is capable ofhybridizing with a fragment of the target gene transcript.

RNA may be synthesized either in vivo or in vitro. Endogenous RNApolymerase of the cell may mediate transcription in vivo, or cloned RNApolymerase can be used for transcription in vivo or in vitro. Fortranscription from a transgene in vivo or an expression construct, aregulatory region (e.g., promoter, enhancer, silencer, splice donor andacceptor, polyadenylation) may be used to transcribe the RNA strand (orstrands); the promoters may be known inducible promoters such asbaculovirus. Inhibition may be targeted by specific transcription in anorgan, tissue, or cell type. The RNA strands may or may not bepolyadenylated; the RNA strands may or may not be capable of beingtranslated into a polypeptide by a cell's translational apparatus. RNAmay be chemically or enzymatically synthesized by manual or automatedreactions. The RNA may be synthesized by a cellular RNA polymerase or abacteriophage RNA polymerase (e.g., T3, T7, SP6). The use and productionof an expression construct are known in the art (see, for example, WO97/32016; U.S. Pat. Nos. 5,593,874; 5,698,425; 5,712,135; 5,789,214; and5,804,693; and the references cited therein). If synthesized chemicallyor by in vitro enzymatic synthesis, the RNA may be purified prior tointroduction into the cell. For example, RNA can be purified from amixture by extraction with a solvent or resin, precipitation,electrophoresis, chromatography, or a combination thereof.Alternatively, the RNA may be used with no, or a minimum of,purification to avoid losses due to sample processing. The RNA may bedried for storage or dissolved in an aqueous solution. The solution maycontain buffers or salts to promote annealing, and/or stabilization ofthe duplex strands.

Preferably and most conveniently, dsRNAi can be targeted to an entirepolynucleotide sequence set forth herein. Preferred RNAi molecules ofthe instant invention are highly homologous or identical to thepolynucleotides of the sequence listing. The homology may be greaterthan 70%, preferably greater than 80%, more preferably greater than 90%and is most preferably greater than 95%.

Fragments of genes can also be utilized for targeted suppression of geneexpression. These fragments are typically in the approximate size rangeof about 20 nucleotides. Thus, targeted fragments are preferably atleast about 15 nucleotides. In certain embodiments, the gene fragmenttargeted by the RNAi molecule is about 20-25 nucleotides in length. In amore preferred embodiment, the gene fragments are at least about 25nucleotides in length. In an even more preferred embodiment, the genefragments are at least 50 nucleotides in length.

Thus, RNAi molecules of the subject invention are not limited to thosethat are targeted to the full-length polynucleotide or gene. Geneproduct can be inhibited with an RNAi molecule that is targeted to aportion or fragment of the exemplified polynucleotides; high homology(90-95%) or greater identity is also preferred, but not necessarilyessential, for such applications.

In another aspect of the invention, the dsRNA molecules of the inventionmay be introduced into cells with single stranded (ss) RNA moleculeswhich are sense or anti-sense RNA derived from the nucleotide sequencesdisclosed herein. Methods of introducing ssRNA and dsRNA molecules intocells are well-known to the skilled artisan and includes transcriptionof plasmids, vectors, or genetic constructs encoding the ssRNA or dsRNAmolecules according to this aspect of the invention; electroporation,biolistics, or other well-known methods of introducing nucleic acidsinto cells may also be used to introduce the ssRNA and dsRNA moleculesof this invention into cells.

As used herein, the term “administration” or “administering” refers tothe process of delivering an agent to a patient, wherein the agentdirectly or indirectly suppresses lrba function and inhibits the growthof tumors. The process of administration can be varied, depending on theagent, or agents, and the desired effect. Administration can beaccomplished by any means appropriate for the therapeutic agent, forexample, by parenteral, mucosal, pulmonary, topical, catheter-based, ororal means of delivery. Parenteral delivery can include for example,subcutaneous intravenous, intramuscular, intra-arterial, and injectioninto the tissue of an organ, particularly tumor tissue. Mucosal deliverycan include, for example, intranasal delivery. Oral or intranasaldelivery can include the administration of a propellant. Pulmonarydelivery can include inhalation of the agent. Catheter-based deliverycan include delivery by iontropheretic catheter-based delivery. Oraldelivery can include delivery of a coated pill, or administration of aliquid by mouth. Administration can generally also include delivery witha pharmaceutically acceptable carrier, such as, for example, a buffer, apolypeptide, a peptide, a polysaccharide conjugate, a liposome, and/or alipid. Gene therapy protocol is also considered an administration inwhich the therapeutic agent is a polynucleotide capable of accomplishinga therapeutic goal when expressed as a transcript or a polypeptide intothe patient.

As used herein, the term “biological activity” with respect to thenucleotides and polypeptides of the subject invention refers to theinhibition of tumor cell growth or proliferation. Thus, cell-basedassays can be utilized to determine whether an agent, such as nucleotideor polypeptide, can be utilized to carry out the method of tumor growthinhibition of the subject invention, as shown in FIGS. 18A-21D.

The term “means for inhibiting or suppressing lrba function” comprisesgenetic and non-genetic means for inhibiting or suppressing lrbafunction. Among the genetic constructs inhibiting lrba function arevarious “gene delivery vehicles” known to those of ordinary skill in theart, that facilitate delivery to a cell of, for example, a codingsequence for expression of a polypeptide, such as an lrba inhibitor, ananti-sense oligonucleotide, an RNA aptamer capable of inhibiting lrbafunction, or other genetic construct capable of inhibiting lrba functionat the transciption, translation, or post-translation level. Methods ofgene silencing and/or knock-down, as described herein, and as known tothose of ordinary skill in the art, can be utilized to suppress lrbafunction, for example. For example, gene therapy comprisingadministration of a dominant negative lrba mutant can be utilized tocarry out the method of tumor inhibition of the subject invention.

Among the non-genetic means for inhibiting lrba function arepharmaceutical agents, or pharmaceutically acceptable salts thereof,which are preferably administered in a pharmaceutically acceptablecarrier.

The term “patient”, as used herein, refers to any vertebrate species.Preferably, the patient is of a mammalian species. Mammalian specieswhich benefit from the disclosed methods of treatment include, and arenot limited to, apes, chimpanzees, orangutans, humans, monkeys;domesticated animals (e.g., pets) such as dogs, cats, guinea pigs,hamsters, Vietnamese pot-bellied pigs, rabbits, and ferrets;domesticated farm animals such as cows, buffalo, bison, horses, donkey,swine, sheep, and goats; exotic animals typically found in zoos, such asbear, lions, tigers, panthers, elephants, hippopotamus, rhinoceros,giraffes, antelopes, sloth, gazelles, zebras, wildebeests, prairie dogs,koala bears, kangaroo, opossums, raccoons, pandas, hyena, seals, sealions, elephant seals, otters, porpoises, dolphins, and whales.

The terms “lrba”, “LRBA”, and “Lrba” (italicized and unitalicized) areused herein interchangeably to refer to the LPS-responsiveCHS1/beige-like gene or its polypeptide product, and includes lrbahomologs (such as human and mouse orthologs), unless otherwise noted.

The terms “comprising”, “consisting of”, and “consisting essentially of”are defined according to their standard meaning and may be substitutedfor one another throughout the instant application in order to attachthe specific meaning associated with each term.

Materials and Methods

Murine RNA Isolation and cDNA Synthesis. Total RNA was prepared usingthe RNEASY kit (QIAGEN, Valencia, Calif.). Poly(A)⁺ RNA was preparedusing the FAST TRACK mRNA isolation kit (INVITROGEN, Calsbad, Calif.).RNA was prepared from murine cell lines as well as liver and thymus ofC57BL6/J mice per the manufacturers' instructions. RNAs were treatedwith Rnase-free Dnase I (AMERSHAM PHARMACIA BIOTECH, Piscataway, N.J.)at 10 U/μg of RNA for 30 minutes at 37° C. to destroy genomic DNA.First-strand cDNA synthesis was primed with random DNA hexamers oroligo(dT) primers at 42° C. for 1 hour using the SUPERSCRIPT II RNase HReverse Transcriptase cDNA Synthesis System (Life Technologies, Inc.,Rockville, Md.).

Cloning and Sequencing of Murine lrba Gene cDNAs. Primers(5′AGAGAAGAGGAGAAGATGTGTGATC3′ (SEQ ID NO. 40); and5′CCAGGCTCCATGCTTGTCTGTGAG3′ (SEQ ID NO. 41) forward and reverse,respectively) were designed from a 143 bp cDNA fragment obtained fromprevious gene-trap experiments (Kerr, W. G. et al. Proc. Natl. Acad. ofSci. USA, 1996, 93:3947) and combined with Lambda GT10 forward andreverse primers (5′AGCAAGTTCAGCCTGGTTAAGT3′ (SEQ ID NO. 42) and5′TTATGAGTATTTCTTCCAGGG3′ (SEQ ID NO. 43), respectively) to amplify thelrba gene cDNA from a mouse B lymphocyte cDNA library (Mouse lymphocyte5′ stretch cDNA library, CLONTECH, Palo Alto, Calif.). These PCRproducts were then cloned and sequenced. New primers were then designedfrom these sequences and further RT-PCR reactions were carried out toextend the cDNA sequence to the 5′ or 3′ direction. The SMART RACEamplification kit (CLONTECH, Palo Alto, Calif.) was used to amplify 5′cDNA ends using the following lrba-specific primers:5′ACTGCAGCAAGCTCCTCCTGTTTTCTC3′ (SEQ ID NO. 44) and a nested primer:5′TGGGCGAAGAGCGGAAACAGAAC3′ (SEQ ID NO. 45), while for 3′ cDNA clonesthe following primers were used: 5′AGAGAAGAGGAGAAGATGTGTGATC3′ (SEQ IDNO. 40) and a nested primer: 5′GAGTGATGGATGATGGGACAGTGGTG3′ (SEQ ID NO.46). PCR conditions for the 5′-RACE and 3′-RACE were as follows usingthe ADVANTAGE polymerase mix (CLONTECH, Palo Alto, Calif.): 94° C. for30 seconds, followed by 5 cycles at 94° C. for 30 seconds, 70° C. for 30seconds, and 72° C. for 3-5 minutes; 5 cycles at 94° C. for 30 seconds,68° C. for 30 seconds, and 72° C. for 3-5 minutes; 20 cycles at 94° C.for 30 seconds, 65° C. for 30 seconds, and 72° C. for 3-5 minutes; and afinal extension at 72° C. for 30 minutes. After the full-length cDNAsequence of the lrba gene was obtained, several primers were designed toamplify the region of the lrba gene cDNA containing its major openreading frame (ORF). The region containing the major ORF of the lrbagene was then amplified from a single source of C57BL6/J liver mRNA andresequenced to confirm that the lrba cDNAs obtained from liver cellswere identical to that amplified from the aligned cDNA fragmentsamplified from primary and transformed B lymphocytes, indicating thatthese represent the major mRNAs expressed from the lrba locus. AllRT-PCR and RACE products were isolated and purified from agarose gelsusing the QIAEX II Gel Extraction Kit (QIAGEN; Valencia, Calif.). Thepurified products were sequenced directly to avoid detecting themutations introduced during PCT. Both strands of each template weresequenced and the sequence was confirmed by sequence analysis of atleast two independent PCR products. PCR products and RACE products werecloned into PCRII vector (TA cloning kit; INVITROGEN, Carlsbad, Calif.)and multiple clones were sequenced. Plasmids were purified from liquidcultures using the QIAGEN plasmid Maxi preparation kit (QIAGEN;Valencia, Calif.).

Human lrba cDNA Cloning and Sequencing. A search of GENBANK indicatedthat the murine lrba gene has a high degree of homology to a 7.3 kbhuman partial cDNA sequence (GENBANK accession numbers M83822) calledBGL (Feuchter, A. E. et al. Genomics, 1992, 13:1237), which was therebytentatively identified as possibly a small fragment of a human lrbagene. The 5′ end of the human lrba gene was obtained by using a 5′primer (5′GCCACCTCCGTCTCGCTGC3′ (SEQ ID NO. 47)) from the mouse lrbagene cDNA sequence and a 3′ primer (5′GGGCACTGGGGAGAATTTCGAAGTAGG3′ (SEQID NO. 48)) from the human BGL sequence. Human lung, brain, and kidneycDNA libraries (MARATHON cDNA Libraries, CLONTECH, Palo Alto, Calif.)were used as templates for the amplification of the 5′ and 3′ ends ofthe human cDNA under the following PCR conditions: 35 cycles at 95° C.for 45 seconds; 60° C. for 15 seconds; 72° C. for 3 minutes. The PCRproducts were cloned into a TA cloning vector and multiple clones weresequenced. Additional PCRs were carried out with the primers from the 3′cDNA clones obtained as described above to complete the sequence of thehuman lrba cDNA. The primer pairs used for these additional 3′ cDNAclones were 5′TTCAGGCAGTTTTCAGGACCCTCCAAG3′ (SEQ ID NO. 49) and5′TAGTGTCTGATGTTGAACTTCCTCCTG3′ (SEQ ID NO. 50). Overlapping regions ofthe 5′ and 3′ human lrba cDNAs were compared and merged with the humanBGL cDNA in GENBANK to construct, for the first time, a completesequence for the human lrba gene (GenBank accession number AF216648).The human lrba gene encodes a 319KD protein that has 2863 amino acids.The amino acid homology between the human and murine lrba gene is 93%(identity 89%, similarity 4%). Like the murine lrba gene, the human lrbagene contains BEACH domain, five WD40 repeats and two novel domains thatare defined as followed (FIGS. 9 and 10).

Northern Blot Analysis. 70Z/3 B lymphoma cells were maintained inRPMI1640 supplemented with 10⁻⁵M 2-mercaptoethanol and 10% fetal bovineserum (FBS). J774 cells were maintained in DMEM supplemented with 10%FBS. 70Z/3 cells were stimulated with 10 ng/ml LPS (Sigma, St. Louis,Mo.) and J774 cells were stimulated with 1 ng/ml LPS for 20 hours.Poly(A)⁺ RNA was prepared from 10⁸ stimulated or unstimulated cellsusing the FASTRACK isolation kit (INVITROGEN, Carlsbad, Calif.).Poly(A)⁺ RNA (5 μg/lane) was size-fractionated by electrophoresis on a6% formaldehyde/1% agarose gel buffered with MOPS, transferred to anylon membrane (STRATAGENE, La Jolla, Calif.) by capillary action in 20×SSC and immobilized by UV cross-linking. The filter was probed with auniformly labeled ³²P probe using the READY-TO-GO DNA labeling kit(AMERSHAM PHARMACIA BIOTECH, Piscataway, N.J.). The probe corresponds toa 2.5 kb PCR product that spans nucleotides 3545-6040 of the murine lrbacDNA. The filter was hybridized with the probe in 2× SSC, 0.5% SDS, 5×Denhardt's containing 100 μg/ml heat denatured salmon sperm DNA at 68°C. overnight. Filters were washed 2 times for 5 minutes at roomtemperature in 2× SSC/0.5% SDS and 2 times for 30 minutes at 68° C. in0.1× SSC/0.1% SDS. Hybridization signals were detected and quantitatedusing a Molecular Dynamics PHOSPHORIMAGER and IMAGEQUANT software.

RT-PCR Analysis of lrba Expression. The cell lines (70Z/3, BAL17, A20,WEHI231, and S194) used for the RT-PCR were obtained from ATCC(Rockville, Md.). Spleen, brain, lung, and bone marrow were obtainedfrom C57BL6/J mice. The preparation of total RNA and cDNA synthesis werecarried out as described above. First strand cDNA reaction products (2μl) were amplified in a 25 μl PCR reaction using primers that detectthree of the lrba isoforms (“5′GGCACAACCTTCCTGCTCAC3′” (SEQ ID NO. 51)and “5′CCTGTCCCCCATTTGAACCC3′” (SEQ ID NO. 52) for the α form:“5′ACGGCTGCTTCTGCACCTTC3′” (SEQ ID NO. 53) and“5′TTTTGGGACAGGGCTTCTCTG3′” (SEQ ID NO. 54) for the β form;“5′GGCACAACCTTCCTGCTCAC3′” (SEQ ID NO. 55) and“5′GCAGATGCTCTCCTCGCTCC3′” (SEQ ID NO. 56) for the γ form). The cyclingprogram was: 94° C. for 30 seconds, followed by 5 cycles at 94° C. for30 seconds, 70° C. for 30 seconds, and 72° C. for 4 minutes; 5 cycles at94° C. for 30 seconds, 68° C. for 30 seconds, and 72° C. for 4 minutes;30 cycles at 94° C. for 30 seconds, 62° C. for 30 seconds, and 72° C.for 4 minutes; and a final extension at 72° C. for 10 minutes.

Gene and Protein Structure Prediction. Analyses of the nucleotide andamino acid sequences for the murine and human lrba gene were performedusing MACVECTOR (Oxford Molecular Group Inc., Oxford, UK). Nucleotidesequence alignments and other analyses were carried out using BLAST(Altschul, S. F. and E. V. Koonin Trends in Biochemical Sciences, 1998,23:444). SMART (Schultz, J. et al. Nucleic Acids Res., 2000, 28:231),and CLUSTLX (Thompson, J. D. et al. Clinical Orthopaedics & RelatedRes., 1997, 241) were used for protein secondary structure predictions.For WD repeat prediction, an algorithm developed by Neer et al (Neer, E.J. and T. F. Smith Cell, 1996, 84:175; Garcia-Higuera, I. et al.Biochemistry, 1996, 35:13985; Neer, E. J. et al. Nature, October 1994,371(6500):812; Smith, T. F. et al. Trends Biochem., 1999, 24:181; Neer,E. J. and T. F. Smith Proc. Natl. Acad. Sci. USA, 2000, 97:960) is used.

Construction, Expression, and Fluorescence Microscopy of the Lrba-GFPFusion Protein. A region from the murine lrba cDNA that includes theBEACH and the WD domains 3′ to the BEACH domain was inserted “in-frame”and upstream of the coding region of a modified GFP gene cloned in amammalian expression vector pEGFP-N2 (CLONTECH, Palo Alto, Calif.).Recombinant clones (called pBWEGFP) were picked, plasmid DNAs preparedand sequenced to confirm that no mutations were introduced during thesemanipulations. Murine 3T3 cells, the macrophage RAW264.7 cells, andhuman 293 cells were transfected by the FUGEN transfection kit (ROCHEMolecular Biochemicals, Indianapolis, Ind.) or by electroporation (GenePulser; BIO-RAD Laboratories, Hercules, Calif.) with 20 μg of linearizedrecombinant plasmid pBWEGFP DNA as well as the control vector pEGFP at250V, 500 μF. One day later, cells were cultured in DMEM containing 0.8μg/ml of G418 (LIFE TECHNOLOGIES, Inc., Rockville, Md.). This medium waschanged every day for the first four days. The surviving G418 resistantcolonies were isolated and used for further experimentation. Forsubcellular localization, cells were plated in glass-covered plates at2.5×10⁵ cells/ml in 2 ml DMEM media with or without LPS at 100 ng/ml.After 12 hours, cells were directly examined by fluorescence microscopyusing a fluorescein isothiocyanate filter to detect expression of GFPfusion proteins. Fluorescent photomicrography was performed using Nikonphotomicrographic equipment model H-III and image software (NIKON,Tokyo, Japan).

Confocal Laser Scanning Microscopy. The RAW 264.7 cells stablytransfected with the pBWEGFP construct were grown on glass coverslipsand stimulated with 100 ng/ml LPS for 24 hours. Golgi and lysosomes werespecifically labeled with BODIPY TR ceramide and LysoTracker Red DND-99(MOLECULAR PROBE, Eugene, Oreg.), respectively, following themanufacturer's protocols. Briefly, for Golgi labeling, cells were washedwith PBS three times and incubated for 30 minutes at 4° C. with 5 μMBODIPY TR ceramide, rinsed several times with ice-cold medium, and thenincubated in fresh medium at 37° C. for another 30 minutes. For lysosomelabeling, medium was changed with pre-warmed fresh medium containing60-75 nM lysosome probe and the cell sample was incubated for 30minutes. Finally, the medium was removed, washed with PBS three times,fixed with 3.7% formaldehyde for 10-20 minutes, washed again, and theslides were mounted with DAPI-containing VECTASHIELD medium (VECTORLABORATORIES, Burlingame, Calif.). Cells were observed on a Zeissinverted Axiovert 100 M laser scanning confocal microscope. Fluorescenceof GFP was excited using a 458/488 nm argon/krypton laser, and emittedfluorescence was detected with 505-530 nm band pass filter. ForLysoTracker Red and BODIPY TR, a 633-nm helium/neon laser was used forexcitation, and fluorescence was detected with a 585 nm band passfilter, using a 100× oil immersion lens. The co-localization function ofLSM510 software (EMBO Laboratory) allows for a reliability of 99% foractual pixels with both fluorophores. The co-localization mask pixelswere converted to white color for clarity.

Immunoelectron Microscopy. The RAW 264.7 cells stably transfected withthe pBWEGFP construct were grown in the presence of 100 ng/ml LPS for 24hours, washed with PBS three times, fixed with 2% paraformaldehyde inphosphate buffer for 1 hour and 4° C., and processed for postembeddingimmunocytochemistry. The cells were scraped from the dishes they weregrown in and pelleted by low speed centrifugation. The pellets weredehydrated in a graded series of ethanol dilutions and embedded ingelatin capsules in LR White resin. The resin was polymerized for 48hours at 50° C. Ultrathin sections of LR White embedded cells werecollected on nickel grids and immunolabeled according to the techniqueof Haller et al. (Haller, E. M. et al. J. Histochem Cytochem, 1992,40:1491) with rabbit-anti-GFP (CLONTECH, Palo Alto, Calif.) at 1:20ration for 1 hour at room temperature, followed by extensive rinsing andthen labeling with 10 nm goat-anti-rabbit IgG-gold (AURION, Wageningen,The Netherlands) for 1 hour at room temperature. Control grids werelabeled by replacing the primary antibody with normal rabbit serum.After extensive washing, thin sections were stained with uranyl acetateand lead citrate before examination with EM.

Primers. The gene-specific primers were designed from the partialsequences of the human lrba that were obtained and from BGL sequence inthe GenBank (GenBank accession numbers M83822). The sequences ofsynthetic oligonucleotides used for PCR amplification were as follows:cdc415mar2: CACACAGAGCATTGTAGCAAGCTCCTC (SEQ ID NO. 57); h65-56153:TGCAGACTTGAAGATTCCG (SEQ ID NO. 58); 3CDS:5′-AAGCAGTGGTATCAACGCAGAGTACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3′ (SEQ IDNO. 59); h6439: GAGTGATGGATGATGGGACAGTAGTG (SEQ ID NO. 60); cdc415mar1:GGGCACTGGGGAGAATTTCGAAGTAGG (SEQ ID NO. 48); and h5end65′:CGAGAAGATGAGAAGATGTGTGATC (SEQ ID NO. 61).

Human RNA isolation and cDNA synthesis. Total RNA was prepared using theRNeasy kit (QIAGEN, Valencia, Calif.). RNA was prepared from cell linesas well as human prostate tumor tissues and normal adjacent tissue perthe manufacturers' instructions. First-strand cDNA synthesis was primedwith gene-specific primers or oligo(dT) primers at 42° C. for 1 h-2 husing the SUPERSCRIPT II RNase H Reverse Transcriptase cDNA SynthesisSystem (Life TECHNOLOGIES, Inc., Rockville, Md.) or PowerScript ReverseTranscriptase (CLONTECH, Palo Alto, Calif.).

5′-RACE, 3′-RACE and the Cloning of human lrba Gene cDNAs. 5′-RACE,3′-RACE of hlrba gene were carried out by using the SMART RACEamplification kit (CLONTECH, Palo Alto, Calif.) and the followingcondition: 5′-RACE:cdc415mar2 as reverse transcription primer, 1-2.5 μgRNAs were used. cdc415mar1 was used for first PCR reaction, h65-56153 () was used for nested primer; 3′-RACE: 3CDS from the kit was used asreverse transcription primer. h5end65′ was used for first PCR reactionand h6439 was used for nested PCR primer. The PCR parameters are: 94° C.for 30 seconds, followed by 5 cycles at 94° C. for 30 s, 70° C. for 30s, and 72° C. for 3-5 min; 5 cycles at 94° C. for 30 s, 68° C. for 30 s,and 72° C. for 3-5 min; 25 cycles at 94° C. for 30 s, 65° C. for 30 s,and 72° C. for 3-5 min; and a final extension at 72° C. for 10 min. AllRT-PCR and RACE products were isolated and purified from agarose gelsusing the QIAEX II Gel Extraction Kit (QIAGEN; Valencia, Calif.). Thepurified products were sequenced directly to avoid detecting themutations introduced during PCR. Both strands of each template weresequenced and the sequence was confirmed by sequence analysis of atleast two independent PCR products. PCR products and RACE products werecloned into PCRII vector (TA cloning kit; INVITROGEN, Carlsbad, Calif.)and multiple clones were sequenced.

Mapping of the 5′ end of the human lrba gene. The 5′ end of the humanlrba gene were determined by SMART 5′ RACE (Clontech, Palo Alto, Calif.)in tumor tissues and adjacent tissues from prostate, human lungcarcinoma, B-cell lymphoma and B-cell lymphoma (AMBION, Austin, Tex.).cdc415mar1 as reverse transcription primer were used. The lrbagene-specific primer cdc415mar2 was used to prime reverse transcriptionusing 1-2.5 μg RNAs. Then first PCR reaction was performed usinggene-specific primer cdc415mar2, h65-56153 was used for nested primer.Products were sequenced both directly and indirectly by first cloninginto pCR2.1 vector (TA cloning kit; INVITROGEN, Carlsbad, Calif.).

Multiple Sequence Alignment. All amino acid sequences were obtained fromthe SWISS-PROT/TrEMBL database at the Expasy web site (www.expasy.ch).Homologous sequences were searched for using the BLAST server of Expasy.To gather tetraspanin and tetraspanin-like sequences from the data base,BLAST searches were performed using a number of sequences from wellestablished members of the tetraspanin superfamily (i.e. CD81, CD82,CD9, CD53, CD63, UPK, RDS, and ROM). A multiple sequence alignment wasinitially achieved with the CLUSTAL1X software. The alignment was thenimproved manually using the GENEDOC software.

Secondary Structure Prediction. To predict the secondary structure ofthe HSH domain, two methods (available on the World Wide Web) based on aconsensus assignment were used. The first method, Jpred², takes amultiple sequence alignment as input and performs a consensus average ofnine different alignment-based secondary structure prediction methods.Alignment-based prediction methods have been demonstrated to have asignificantly better accuracy than those using single sequences, andconsensus averaging by Jpred² has been shown to increase the accuracy to72.9%. The use of alignment-based secondary structure prediction methodsrequires the sequences to have a degree of homology of at least ˜25%.

RT-PCR Analysis of hlrba Expression. The cell lines MCF7 breast cancercell line, 293 cell line, pre-B (6417); Raji B cells; HTB4 lung cancer;H322 human lung cancer; A539 human lung cancer used for the RT-PCR wereobtained from ATCC (Rockville, Md.). The preparation of total RNA andcDNA synthesis were carried out as described above. First strand cDNAreaction products (2 μl) were amplified in a 25 μl PCR reaction usingprimers.

Following examples illustrate procedures for practicing the invention.These examples should not be construed as limiting. All percentages areby weight and all solvent mixture proportions are by volume unlessotherwise noted.

EXAMPLE 1 Cloning and Sequencing of the Murine lrba cDNA

An LPS-inducible gene was identified by integration of Gensr1 gene-trapretrovirus (Kerr, W. G. et al. Proc. Natl. Acad. of Sci. USA, 1996,93:3947). A partial cDNA sequence of the LPS-inducible gene-trap cellclone, 7a65, was used to design PCR primers to amplify the upstream anddownstream regions of cDNA from a mouse B lymphocyte library. Initially,a 1.6 Kb cDNA sequence was obtained by this strategy. Sequence analysisconfirmed that this 1.6 Kb cDNA sequence contains the original 142 bpsequence obtained by gene-trapping (Kerr, W. G. et al. Proc. Natl. Acad.of Sci. USA, 1996, 93:3947). 5′ RACE reactions using anti-sense primersfrom the 5′ end of this 1.6 Kb region yield additional 5′ cDNA sequencesincluding the 5′ UTS of the lrba gene as well as the ATG of its majorORF. Sense strand primers were also designed from the 1.6 Kb cDNAsequence and three 3′ RACE fragments of 2.5 Kb, 2 Kb, and 1.4 Kb wereobtained that have identical 5′ end sequence; however, their 3′ endsdiffer substantially. The amino acid sequence of the major ORF in themurine lrba cDNA is shown in FIG. 1A. The human lrba orthologue isobtained as described in the Experimental Procedures section.

Sequence analysis of the lrba cDNAs indicated the existence of threeisoforms with identical 5′ ends that differ at their 3′ termini. Theseisoforms include a 9903 bp form (lrba-α), a 9396 bp form (lrba-β) and8854 bp form (lrba-γ) encoding proteins of 2856, 2792, and 2779aa,respectively. All three ORFs begin with the same Kozak consensus ATG atnucleotide 308. The first 2776aa of the β form are identical to thefirst 2776aa of the α form, while the 16aa at its C-terminus are uniqueto it. The first 2769aa of the γ form are identical to the first 2769aaof the α and β forms with its C-terminal 10aa unique to it; the α formhas its C-terminal 80aa unique to it (FIG. 1). Homology search indicatesthat all lrba isoforms have a BEACH domain (Nagle, D. L. et al NatureGenetics, 1996, 14:307); however, the lrba-α isoform has 5 WD repeats,lrba-β has 3 WD repeats while lrba-γ lacks WD repeats (FIG. 1B). Theisoform specific unique coding sequences and the associated 3′untranslated sequence (totally 1267 bp for α form, 761 bp for β form,and 845 bp for γ form) show no significant homology with each other.Interestingly, only the α form has an AATAAA sequence for polyArecognition and a TGA stop codon, while the β and γ forms have TAA stopcodons.

EXAMPLE 2 Lrba Orthologues Exist in Diverse Organisms and Belong to aNovel Gene Family

Homology analysis revealed that lrba has significant homology with thepartial protein sequence DAKAP550 (Han, J. D. et al. Jour. Biol. Chem.,1997, 272:26611), which is an AKAP, and with AKAP550 (GENBANK accessionnumber AAF46011) predicted from the Drosophila genomic sequence (GENBANKaccession number AE003433). A longer sequence for this gene is predictedfrom the genomic sequence and is designated dLRBA, which is identical toAKAP550 except that it has an additional 160aa at its N-terminus. Asused herein, the first letter of the genus is placed before the gene'sname to distinguish the lrba genes of different species. Thus, DAKAP550is a partial sequence of dLRBA and AKAP550. Amino acid alignmentanalysis shows that the murine LRBA protein has 85% aa identity withhuman LRBA, 51% aa identity with dLRBA and 35% aa identity with the C.elegans CDC4L gene (GENBANK accession number T20719) (designated cLRBAfor clarity) (FIG. 1B). This homology analysis shows that the lrba andDAKAP550 genes are othologues based on their high homology that extendsfrom their N terminus to the C terminus (FIGS. 1-3 and Table 1).Furthermore, two putative PKA binding sites are found in all lrbaorthologues (FIGS. 2A and 2B) and are structurally similar to the B1 andB2 RII binding sites of DAKAP550, a protein that has been demonstratedto bind PKA in vitro and in vivo (Han, J. D. et al. Jour. Biol. Chem.,1997, 272:26611 ). This region is highly conserved in lrba orthologuesin mice, man, Drosophila, and C. elegans (FIG. 2A) and potentiallyprovides another two PKA binding sites for DAKAP550. Unexpectedly, theB1 and B2 sites of DAKAP550 are not found in other LRBA proteins; theymay be species-specific and these potential RII binding sites need to beconfirmed by biochemical studies.

TABLE 1 Identities Positives Length (aa) mLBA dLBA  92-405  47-394 51%73% 314  405-959  601-1160 55% 75% 555  998-1576 1542-2127 36% 53% 5791793-2856 2642-3727 56% 74% 1064 mLBA cLBA  65-946  164-1057 42% 61% 8821300-1571 1065-1333 39% 59% 271 1787-2856 1436-22512 47% 64% 1070 mLBAhLBA   1-2856   1-2863 85% 88% 2856 mLBA mBG 1934-2839 1460-2335 27% 43%906 mLBA hFAN 2038-2841  163-913 29% 45% 803

Table 1 shows the protein homology between LRBA and dLRBA, mBG, andHFAN, showing the percentage of identity, and positive gaps. Thepositions of each fragment are also indicated.

These lrba orthologues also have a highly conserved long C-terminalregion (around 1000 amino acids) shared with a group of proteinsincluding CHS1/BG (Perou, C. M. et al. Nature Genetics, 1996, 13:303;Kingsmore, S. F. et al. Jour. Invest. Med., 1996, 44:454), FAN(Adam-Klages, S. et al. Cell, 1996, 86:937), LVSA (Kwak, E. et al. Cell,1999, 10:4429) proteins (FIGS. 2A and 2B), and a number of anonymousORFs. They constitute a new gene family. The conserved region containsan unidentified region followed by one BEACH domain and several WDrepeats. Several WD repeats are found in the unidentified region ofhomology in these genes when about 1000 aa of C-terminal sequence issearched for WD repeats; however, no WD repeat is predicted when thisregion is analyzed alone (data not shown). Thus, this region isdesignated herein as WD repeat-like domain (WDL). In aggregate, and notto be limited by theory, the entire WDL-BEACH-WD (WBW) structure mayhave a precise functional role since the WD repeats found in the WBWstructures of different beige-like genes have a higher degree ofhomology with each other than with other WD repeats in proteins thatlack a BEACH domain (FIG. 3). This homology analysis suggests theevolutionary conservation of the WBW structure in a gene family thatincludes lrba, chs1/beige, FAN, lvsA, and other unidentified ORFs inGENBANK. However, the BEACH domain can exist without WD motifs as in thecase of lrba-γ (FIGS. 1A, 1B and 3). It is shown herein that all BEACHdomains have an SH3 binding site (consensus sequence PXXP), an SH2binding site (consensus sequence YXXhy) (Pawson, T. and J. D. ScottScience, 1997, 278:2075), and a tyrosine kinase phosphorylation site(consensus sequence: (RK)-x(2,3)-(DE)-x(2,3)-Y) (Patschinsky, T. et al.Proc. Natl. Acad. Sci. USA, 1982, 79:973; Hunter, T. J. Biol. Chem.,1982, 257:4843; Cooper, J. A. et al. J. Biol. Chem., 1984, 259:7835), asshown in FIG. 3. These putative binding sites show that WBW proteins mayinteract with multiple signal transduction components.

EXAMPLE 3 Analysis of lrba mRNA Expression

Northern blot analysis indicates a single mRNA of about 10 Kb encodingthe lrba gene is present in LPS-induced J774 macrophages and 70Z/3 Bcells (FIG. 4A), as well as in other B cell lines (WEHI231, BCL1) andthe macrophage cell line, RAW264.7 [RAW267.4]. The size (˜10 Kb) of thetranscript is consistent with the cDNA sequence analysis describedherein (9903 bp for lrba-α). The expression of the lrba gene issignificantly up-regulated in LPS-induced J774 macrophage cells as thelrba mRNA is nearly undetectable in J774 cells in the absence of LPSstimulation. The level of lrba mRNA is increased by 3 fold in 70Z/3 Bcells (FIG. 4A) using β-actin mRNA as an internal standard. Theupregulation of lrba expression in the B cell lines is consistent withthe FACS analysis of lacZ induction in the 7a65 gene-trap cell clone(Kerr, W. G. et al. Proc. Natl. Acad. of Sci. USA, 1996, 93:3947).

A multiplex RT-PCR assay was also developed that can simultaneouslydetect the expression of the lrba mRNA isoforms. RT-PCR analysis of lrbamRNA (FIGS. 4B and 4C) shows that lrba-β mRNA is expressed in all celllines and tissues analyzed; however, lrba-α mRNA is absent in 70Z/3,lung and bone marrow and is less abundant in spleen and lung, suggestingthat these different isoforms may have discrete functions in differenttissues.

EXAMPLE 4 Subcellular Localization of LRBA-GFP Fusion Protein ShiftsUpon LPS Stimulation

All mutations in beige or chs1 genes result in truncated proteins thatlack the BEACH and COOH terminal WD repeats (Certain, S. et al. Blood,2000, 95:979). This region may contain sequences critical to thefunction of chs1/beige and lrba genes. In particular, the ability oftheir gene products to associate with intracellular vesicles toinfluence their trafficking may be lost in these truncated mutants.Therefore, a GFP fusion with the BEACH-WD region of lrba called BW-GFPwas created. As shown in FIGS. 5A-5I, fluorescence microscopy of RAW267.4 cells stably transfected with an expression vector encoding theBW-GFP fusion shows that the BW-GFP protein is present in the cytosolwith rare cells showing a vesicular staining pattern in the absence ofLPS stimulation (FIG. 5A). However, this vesicular staining pattern isdramatically increased in these cells following LPS stimulation (FIG.5B). Both the percentage of cells and the degree of vesicular stainingin each cell are increased following LPS stimulation. RAW267.4 cellsstably transfected with a GFP control construct show no change in theirGFP fluorescence pattern upon LPS stimulation (FIG. 5C).

To determine which vesicular compartments the BW-GFP fusion localizesto, the RAW264.7 cells stably transfected with the pBWEGFP constructstained with a lysosome specific dye (FIG. 5E) and trans-Golgi specificdye (FIG. 5H) were analyzed with confocal microscopy. The mergedpictures show that some LRBA-GFP proteins are co-localized withlysosomes (FIG. 5F, white area) and co-localization with the trans-Golgicomplex (FIG. 5I, white peri-nucleus area).

Immunogold labeling experiments were also performed that show theLRBA-GFP fusion protein can be found in association with the Golgicomplex (FIG. 6D), lysosomes (FIGS. 6B and 6F), endoplasmic reticulum(FIG. 6C), plasma membrane (FIG. 6E), perinuclear ER (FIG. 6E), andendocytic vacuole (FIG. 6A, as the gold particles are labeling aclathrin coated endocytic vacuole, which indicates that it is involvedin endocytosis and not exocytosis). The immunoelectron microscopyresults agree well with the observations made by fluorescence microscopyand confocal fluorescence microscopy.

EXAMPLE 5 Exon/Intron Structure of the Human lrba Gene

The genomic locus of lrba gene is composed of 58 exons and 57 introns,spinning over a 700 K bps genomic sequence. Exon 1 and exon 2 containthe first part of the 5′ UTR, exon 2 contains the rest of the 5′UTR andthe start methione, while exon 58, the final exon, contains part of theWD5 and the whole 3′UTR. There are two considerably large exons—exon 24(1059 bps) and exon 58 (1148 bps). The entire SET domain is encoded byone exon—exon 24, while other domains are econded by multiple exons. Theremaining exons range in size from 33 to 435 bps, most are below 200bps. All exon/intron junctions conform to the GT-donor/AG-acceptor rule(Breathnach and Chambon, 1981)(Table 1). The function of the lrba geneis defined by its domain structure consisting of BEACH domain, WDrepeats, HSH domain and SET domain and potential RII binding sites. TheBEACH domain is encoded by exons 45 to 51. The 5-WD repeat domain isencoded by exons 54 to 58. Isoforms are formed by splicing with splicingsite inside the exons of the other isoforms.

Table 2 shows the exon/intron organization of the human lrba gene.

TABLE 2 Exon/Intron Organization of the Human lrba Gene Exon Intron ExonNo size (bp) 5′Splice donor SEQ ID NO. Intron No size (kb) 3′Spliceacceptor SEQ ID NO. 1 ~67 AGT ATC TGG gtgaggaag 62 I 0.340 tccaataag GGTTTG GCG 119 2 435 TTT AAC CTG gtaagtcca 63 II 85.572 ccttgtaag TTG GTAGGA 120 3 232 TGA TAG CAG gtatgattt 64 III 0.217 tgtttccag ATC TTT TGG121 4 101 GGA CGA TCG gtaaaaaaa 65 IV 7.224 tcttcatag CCT CCA CAT 122 596 AGT GCT GCA gtaagtaa 66 V 4.458 ttcctttag GCT ATT GCA 123 6 122 TTTGTA TTG gtatgtatt 67 VI 0.089 tctttatag TTT CAG AAC 124 7 127 CCA CAAAAG gtacatgat 68 VII 0.674 cttctgcag TGG TAT ATG 125 8 120 ACT AGC GATgtaagtagt 69 VIII 1.266 cttttacag ACC TTT GAC 126 9 147 GGA TAC AAGgtagtttgc 70 IX 5.537 ttcttagag GGT ACA TTT 127 10 198 ATG CTC CAGgtactaact 71 X 0.192 tcttacaag GAT GTA AAG 128 11 134 GAC TAT ATGgtgagtgcc 72 XI 1.971 aaattctag TTC AAC CTT 129 12 109 CTT GAA AAGgtaaagtat 73 XII 0.306 tttttgcag TCT TCC AAA 130 13 153 CCA GCC AAGgtaatatat 74 XIII 5.619 attctgtag GTT CAA CTG 131 14 169 AAG GAT TAGgtatataat 75 XIV 2.233 ttttaaaag ATG GAC CGC 132 15 80 GTG ATG AAGgtaggttca 76 XV 1.282 tttttgaag GAT TCT GGA 133 16 63 ATG CAT GAGgtaatatat 77 XVI 3.245 tgattatag GAT GAC AAT 134 17 98 TGG GTT ACGgtaagagtt 78 XVII 20.299 ttcattcag TGT TAT CTA 135 18 93 GGC CCC AAAgtaagtatg 79 XVIII 1.209 taattgcag GAG GAA AGC 136 19 109 CTG TTT GAGgtaggaatg 80 XIX 0.738 cttctgtag ATT CTT ATA 137 20 82 AAA CCC CTCgtatgtatg 81 XX 2.220 agattacag AGA TAC TAA 138 21 124 AAA CAG GAGgtaagctga 82 XXI 0.318 aattttcag GAG CTT GCT 139 22 193 CAT TCA AAGgtaagtttc 83 XXII 14.688 ttcacctag GTC ACT TTT 140 23 1059 GTG CTT GAGgtgatttta 84 XIII 0.982 tgtattaag ATA TCA AGG 141 24 179 GTG GAG AAGgtttgtcta 85 XXIV 1.148 tttggacag CCA TTC AAC 142 25 154 TCG GCT ACAgtaaggact 86 XXV 0.423 tctttacag CAT GAA CTG 143 26 181 TCC GAC TAGgtgagctgc 87 XXVI 4.039 aaattacag TTT GTG CAG 144 27 122 GCA GCG AAGgtaagtata 88 XXVII 0.450 cttaaatag AGC CCA GTG 145 28 108 AGA GAC ATAgtaagttac 89 XXVIII 12.124 ttttcccag GAG GAT AGC 146 29 160 CAC TCT CTGgtaagtttg 90 XXIX 3.193 atgatatag AAA TCA CAC 147 30 442 TTT TGA CAGgtactgata 91 XXX 10.928 ttattacag AAG TGT CAT 148 31 134 AAT CAC CAGgtgagttag 92 XXXI 8.713 cttttatag GCA GTA GAT 149 32 79 AAA TAT GAGgtatttaag 93 XXXII 1.909 tttccttag TAT TAC AGA 150 33 134 AAG GAA CAAgtaagtggt 94 XXXIII 7.964 ttaaaatag GTC TGG TTT 151 34 62 TGT TCT CAGgtgagtggc 95 XXXIV 35.939 tttttatag GAG TGG CAA 152 35 65 ATG AGG AAGgtaatttat 96 XXXV 26.429 ttcttacag GTT GCT TAG 153 36 109 GAA TTT GAGgtaggttac 97 XXXVI >28.963 ctctccaag TCA CTG TGT 154 37 167 TGC AGT GAGgtaaaggga 98 XXXVII 83.886  cattgtag TCG TCC TCT 155 38 125 TGG AAC ATGgtcagtgg 99 XXXVIII 1.891 atgttttag TGT GCA TTT 156 39 33 ACA GCA AAGgtaagcatt 100 XXXIX 6.179 tcatttcag CCA CAG ATG 157 40 147 ATC TTG CCGgtaaatttg 101 XXXX 2.515 ttttggcag GTC CTG TTA 158 41 137 GAC CCC AAG gt102 XXXXI 96.572 cctcattag ATC TTG GCA 159 42 118 CAA ACA GAG gtaatgtgt103 XXXXII 3.088 ctgttgtag TTG CTG TGA 160 43 103 TCA AAC CAG gtactgttt104 XXXXIII 15.997 ttcttgcag ACG TAT TTC 161 44 116 CGA TAG CAGgtaacctaa 105 XXXXIV 3.840 ccctatcag GAC GGA GTT 162 45 113 TTG TCC AAGgtaatttct 106 XXXXV 30.846 tattggcag CCA ATA GGA 163 46 141 CTA AGA ATAgtaagttca 107 XXXXVI 1.015 attttttag GAA CCC TTT 164 47 120 GAT ATT AAGgtacagaaa 108 XXXXVII 19.536 tttatatag GAG TTG ATC 165 48 153 AAC AGATTG gtaagataa 109 XXXXVIII 65.358 ttttttcag GCC CTG GAG 166 49 169 TTGAGA GAG gtaa9ttat 110 XXXXIX 24.093 ccttttcag GCT GTT GAA 167 50 90 ATGCAA GTG gtaagtgct 111 XXXXX 4.443 ctcctgcag AGT CCA TTG 168 51 178 ACCTTC CTG gtaagtaaa 112 XXXXXI 5.563 gaattccag CTC ATC AAG 169 52 63 CTCTCA TAG gtctgtcac 113 XXXXXII 5.176 ttcttacag CCA GCA ATA 170 53 156 CAGACA CAG gtaattttc 114 XXXXXIII 7.441 gcattacag GAA GAT TGA 171 54 168ACC CAG GCA gtaagtatg 115 XXXXXIV 16.043 ttcctaaag GTG AGA CTG 172 55102 GTT CAC AAG gtaaacctg 116 XXXXXV 3.286 tcttctcag AAG GAC CAT 173 56197 AAC ATA AGA gtgagtgcc 117 XXXXXVI 4.444 gtctcacag GCC ATC CAG 174 57152 CGA CCA GAG gtaacactg 118 XXXXXVII 12.028 ttctcctag GTG CAT CAT 17558 1148 Total 9936 >716.138

EXAMPLE 6 Molecular Phylogenic Relationship of hlrba Proteins with OtherWBWs

Phylogenic analysis of the WBW family reveals that the members can begrouped into two major families, as shown in FIG. 12. One family iscomposed of proteins from C. elegans, D. melanogaster, H. sapiens, S.pombe, S. cerevisiae, A. thaliana, D. discoideum, and the other familycontains proteins from H. sapiens, M. musculus, Dr. melanogaster, C.elegans, A. thaliana, B. taurus, L. major. These can be furthersub-grouped into five distinct subfamilies, each of which may containsevery species from the very ancient unicellular eukaryote to human. Lrbain human and murine, AKAP550 in fruit fly, F10F2.1 in C. elegans areorthologs as indicated previously, while NBEA and CG1332 are very closeto lrba gene. Lrba, CHS1/beige and FAN belong to the same family.Despite the divergence of these species over several hundred millionyears, there is a high degree of sequence conservation in the BEACHdomain, which may suggest an important role in the life of the cellconcerning the BEACH domain.

EXAMPLE 7 The Human lrbaε Alternative Transcript has Two In-frame ORF

The ORF prediction shows there are two in frame ORFs in the human lrbaεalternative transcript. One ORF encodes a 72 amino acid protein, anotherencodes a 2782 amino acid protein. A very conserved motif (p21 RAS motifIV(LLGVGGFD (SEQ ID NO. 176))) is missing from both proteins as a resultof the disruption. Both ATGs are in the Kozak sequence and thus couldserve as translation initiation sites. According to the translationscanning theory, the translation of the first ORF should not be aproblem. There are three possibilities for the translation of the secondORF. The first possibility is leaking scanning, meaning that someribosomes do not recognize the first ATG, but recognize the later ATG.However, there are four ATGs before the main ATG, and there is a longstem secondary structure between the two ORFs. Therefore, it is unlikelythat the leaking model is the mechanism of translation. The secondpossibility is reading through translation, meaning that the translationmachinery ignores the stop codon and reads through it. However, thereare 10 stop codons between the two ORFs. Likewise, this is unlikely. Athird possibility is that IRES (internal ribosome entry signal)translation is cap-independent. There is no homologous sequence betweenIRES, but they have complex secondary structure, such as long stemsecondary structure. The RNA sequence between the two ORFs of humanlrbaε can form a long stem structure, which could further make theleaking scanning or reading through impossible. Some mRNAs encodingpro-apoptic proteins, including Apaf-1 and DAP5 are also translated viaan IRES element. IRES-independent initiation is sometimes utilizedduring mitosis. The numberous mRNAs whose 5′ UTR structures likelyinterfere with the 5′ cap-dependent ribosome are good candidates for thepresence of an IRES. However, the prediction of an IRES from onlylooking at the 5′ UTR could be strengthened by a better understanding ofthe structural components that comprise these IRES elements.

EXAMPLE 8 Identification of the Five Isoforms of the Human lrba Gene

Four isoforms that encode four different proteins are present in humanlrba gene, among which three isoforms differ at C-terminal: h-lrbaα hasfive WD repeats, h-lrbaβ lacks WD repeats, h-lrbaδ lacks WD repeats andpart of BEACH domain. The fourth isoform h-lrbaγ has a YLLLQ (SEQ ID NO.32) additional sequence between BEACH domain and WD repeats. Thisinsertion isoform also exists in murine LRBA gene, and the 15 bpnucleotide sequence insertion remains unchanged. All the isoforms aresummarized as shown in FIG. 13.

TABLE 3 Pattern of alternative Isoforms Positions Features Implicationssplicing* 1α There is one Disrrupt the Bicistron may exist in Cassetteextra exon coding sequence eukaryotes. Ribosome between Exon2 of thelrba gene Internal entry sequence. and Exon 3 at the N-terminus 2βPoly(A) There is a 312 bp 1. The BEACH domain is Multiple alternativeAlu repeat not a minimum domain, Polyadenylation splicing after sequenceat the could be actually Site Exon 48 5′UTR, splitting composed of twothe BEACH domains. 2. The Alu domain at two sequence may regulate thirdinto two the translation of LRBA potential gene or other gene. domains3γ 15 bp insertion The insertion Leucine (L) is a Retained intron beforeExon encodes a hydrophobic amino acid 51, just after YLLLQ peptide andmay be involved in BEACH insertion into the protein-protein domain andLRBA protein. interaction(as Leucine before WD Zipper structure). Thatrepeats there are three consecutive Ls in a short sequence is unusualand Y could be a potential target for phosphorilation. 4δ Poly(A) Theisoform Although BEACH Multiple alternative doesn't have WD domain andWD repeats Polyadenylation splicing after repeats but often staytogether, they Site Exon 52 BEACH domain are separate domain and canexist and function separately. 5ε An additional Alternative LRBA may usedifferent Multiple exon at 5′ end promoter and promoters to regulate thePromoters (Exon 5′-1) transcription start expression of LRBA. beforeExon 1 site

The LRBA gene and five isoforms of the LRBA gene are disclosed andcharacterized herein. Northern blot experiments show that expression oflrba is upregulated 2-4 fold following LPS stimulation of B cells andmacrophages. A homology search of GENBANK reveals that lrba gene hasothologues in C. elegans, Drosophila, mice and humans and paralogues indiverse species ranging from yeast to human. These genes define a newprotein family that are designated the WBW gene family herein becausethe members share an evolutionarily conserved structure over a longprotein sequence (around 1000 aa). The analysis of subcellularlocalization with a BEACH-WD-GFP fusion protein described hereinprovides the first direct evidence that the lrba member of the WBWfamily can physically associate with various vesicular compartments incells. Furthermore, it is proposed that the lrba gene is also an AKAP,suggesting that WBW family proteins may have microtubule and PKA bindingproperties like AKAPs (Colledge, M. and J. D. Scott Trends in CellBiology, 1999, 9:216). Studies of FAN suggest that WBW proteins can bindto cytoplasmic tails of activated receptors via their WE repeats(Adam-Klages, S. et al. Cell, 1996, 86:937).

The evidence suggests that WBW proteins are involved in intracellularvesicle trafficking. For example, the strikingly enlarged vesicles inbeige/CHS cells occur in membrane-bound organelles. The CHS1/BG proteinhas a similar modular architecture to the VPS15 and Huntington proteinsthat are associated with the membrane fraction (Nagle, D. L. et al.Nature Genetics, 1996, 14:307) and the lvsA gene that is essential forcytokinesis (Kwak, E. et al. Cell, 1999, 10:4429)-a process that alsoinvolves fusion of intracellular vesicles (Jantsch-Plunger, V. and M.Glotzer Curr. Biol., 1999, 9:738; Heese, M. et al. Curr. Opin. PlantBiol., 1998, 1:486). FAN may also be involved in vesicle traffickingsince FAN-deficient mice, after cutaneous barrier disruption, havedelayed kinetics of skin recovery that requires secretion of vesicles(Kreder, D. et al. EMBO Journal, 1999, 18:2472; Elias, P. M. J. Invest.Dermatol., 1983, 80:44s). However, there is no direct evidence thatthese WBW proteins directly associate with vesicles. In contrast, othersfound unexpectedly by Western blot that the BG, LVSA, and DAKAP550proteins are present in the cytosolic fraction of cells and not in themembrane fraction (Kwak, E. et al. Cell, 1999, 10:4429; Perou, C. M. etal. Jour. Biol. Chem., 1997, 272:29790) or cytoskeleton (Han, J. D. etal. Jour. Biol. Chem., 1997, 272:26611). This paradox can be explainedby hypothesizing (without being limited by theory) that these proteinsare not constitutively associated with vesicles, but rather associatewith vesicles under certain conditions like LPS stimulation. Thishypothesis agrees well with the observation that an LRBA-GFP fusionprotein is located in the cytosol; however, it becomes associated withvesicles following activation of the cells by LPS stimulation. Confocalmicroscopy also shows this fusion protein co-localizes with thetrans-Golgi and lysosomes. Immunoelectron microscopy furtherdemonstrates that it is also localized to endoplasmic reticulum and theplasma membrane as well as the trans-Golgi complex and lysosomes.Therefore, it is established herein that the BEACH-WD-GFP fusion proteinis associated with the vesicular system. This may be true for the intactLRBA protein as well as for other WBW proteins like CHS1/BG, LVSA, andFAN, since they share high homology with the region in mouse lrba thatwas used for the GFP fusion experiment. The activation-triggered vesicletrafficking hypothesis is further supported by the following: (1) BEACHdomain contains a tyrosine phosphorylation site, (2) the WD repeatsbinding site of FAN contains a serine residue (Adam-Klages, S. et al.Cell, 1996, 86:937), it is possible that this serine is a target ofserine kinases, as some experiments suggest that the WD repeats bindingrequires phosphorylation of the WD binding sites (Skowyra, D. et al.Cell, 1997, 91:209) and (3) MAPK was suggested to control the movementof lytic granules of NK cells (Wei, S. et al. Jour. Exper. Med., 1998,187:1753). Potentially, WBW protein functions are activated by tyrosineand/or serine/threonine kinases following stimulation by agents likeLPS. Although the GFP fusion experiment previously described does notdemonstrate that the BEACH domain and/or the WD repeats in LRBA directlyassociate with intracellular vesicles, it is proposed that the BEACHdomain binds to vesicles while the WD repeat domains bind to amembrane-associated protein. It is proposed that because BEACH domainsand WD repeats exist separately in some proteins, they have separatefunctions. For instance, the WD repeats of the FAN protein bind to thecytoplasmic tail of the TNFR55 receptor independent of the BEACH domain(Adam-Klages, S. et al. Cell, 1996, 86:937). It is worth noting that theFAN gene is made up almost entirely of the sequence in the highlyconserved WBW structure (FIG. 3), therefore other WBW-containingproteins may act like FAN and bind the cytoplasmic tails of TNFR55 orTNFR55-like receptors.

As indicated above, the lrba gene is a potential AKAP. The recentlycompleted genomic sequence of Drosophila indicates that lrba has anorthologue in Drosophila (DAKAP550) that is capable of binding toprotein kinase A (Han, J. D. et al. Jour. Biol. Chem., 1997, 272:26611).The DAKAP550 gene is expressed in all tissues throughout development andis the principal A-kinase anchor protein in adult flies; it is enrichedin secretory tissues such as neurons and salivary glands, and is foundconcentrated in the apical cytoplasm of some cells (Han, J. D. et al.Jour. Biol. Chem., 1997, 272:26611), in agreement with the proposedfunction in secretion of lrba. Although the B1 and B2 RII binding sitesof DAKAP550 are not present in mLRBA, hLRBA, and cLRBA, two sequencesare disclosed that are very similar to the B1 and B2 RII binding sitesin all lrba orthologues. The two sequences are predicted to form twoadjacent amphipathic helices characteristic of PKA binding sites,satisfying the requirement of the hydrophobic interaction mechanism ofRII peptide binding to the RII subunits of PKA revealed recently(Newlon, M. G. et al. Nat. Struct. Biol., 1999, 6:222). Thus, lrba mayserve as an AKAP that is involved in cAMP-mediated signaling secretoryprocesses by translocating PKA to specific membrane sites. Thistranslocation may require microtubule binding as suggested by the recentfinding that another WBW protein, human CHS1, can associate withmicrotubules (Faigle, W. et al. J. Cell Biol., 1998, 141:1121). Based onthese findings, it is proposed a two-signal model for the function ofthe WBW protein family using the lrba gene as a protoype: LRBA isconstitutively associated with PKA like other AKAPs and following LPSstimulation (signal one) the BEACH domain is phosphorylated. Thisenables the LRBA/PKA complex to bind to intracellular vesicles andtether vesicles to microtubules for transport to the plasma membrane. Atthe membrane, a second signal is required that generates cAMP. Bindingof locally generated cAMP to the LRBA/PKA complex releases PKA, allowingit to phosphorylate cytoplasmic tails of activated receptors to enablebinding of LRBA via its WD repeats. This final step would result invesicle fusion with the plasma membrane (FIG. 7). Many immune processesneed a second signal such as in the case of co-stimulators. Withoutbeing bound by theory, it proposed that a first signal activates animmune cell to transport enough vesicles to the plasma membrane areathat contact another cell. A second signal generated by the contact withthe target cell produces cAMP that stimulates PKA activity resulting inmembrane fusion of vesicles. Thus, LRBA and other WBW proteins mayprovide a means for eukaryotic cells to direct the fusion ofmembrane-bound vesicles in a polarized fashion, in coordination withsignal transduction complexes at the plasma membrane as is required ofmany different effector cell types in the immune system (Stinchcombe, J.C. and G. M. Griffiths Jour. Cell Biol., 1999, 147:1).

Increasing evidence suggests that all clinical symptoms of CHS/beigepatients could be explained by a secretion malfunction. The cytolyticproteins (granzymes A/B and perforin) in CHS CTL are expressed normally,but are not secreted upon stimulation (Baetz, K. et al. Jour. of Immun.,1995, 154:6122). Secretion of other enzymes are also defective inmacrophages and neutrophils (Barak, Y. and E. Nir American Journal ofPediatric Hematology-Oncology, 1987, 9:42) as are the membranedeposition of class II molecules (Faigle, W. et al. J. Cell Biol., 1998,141:1121) and CTL-4 (Barrat, F. J. et al. Proc. Natl. Acad. of Sci. USA,1999, 96:8645). However, there is a dispute over whether giant lysosomesin beige/CHS disease are a result of abnormalities in the fusion orfission of lysosomes (Baetz, K. et al. Jour. of Immun., 1995, 154:6122;Barrat, F. J. et al. Proc. Natl. Acad. of Sci. USA, 1999, 96:8645;Perou, C. M. et al. Jour. Biol. Chem., 1997, 272:29790; Cervero, C. etal. Sangre, 1994, 39:135; Barbosa, M. D. et al. Nature, 1996, 382:262;Menard, M. and K. M. Meyers Blood, 1988, 72:1726). How the secretionpathway is impaired is unclear. The characterization of the lrba geneand the model for its function, described herein, may provide amolecular explanation for these two major cellular dysfunctions ofCHS/beige: giant vesicles and secretion malfunction. Vesicles mayrequire association with the BEACH domain of CHS1 for fission and/ormovement to the plasma membrane. After reaching the plasma membrane,they then require recognition of certain membrane proteins by the WDrepeats to mediate fusion with the plasma membrane. This requires CHS1proteins to be full-length for proper function since the WD repeats areat the COOH terminus. Thus, truncated beige/CHS protein molecules (orperhaps LRBA proteins) that lack the COOH terminal WD repeats would beexpected to cause disease (Certain, S. et al. Blood, 2000, 95:979). Thegiant lysosomes in the affected cells may come from the failure ofvesicle movement and/or fusion with the membrane. Similar disorders ofbeige/CHS have also been described in mink, cattle, cats, and killerwhales. Given the structural similarity of the WBW gene family, it isproposed that the genetic mutations in these species also involve otherWBW genes. There are also other lysosomal trafficking mutants in micewith similar phenotypes to beige that may also involve mutation of otherWBW gene family members.

In summary, the existence of a novel gene family, the WBW family, isdemonstrated herein, which includes the lrba gene that: (1) isassociated with the vesicular system, including the Golgi complex,lysosomes, endoplasmic reticulum, plasma membrane, and perinuclear ER,(2) is LPS inducible, (3) is an A kinase anchor protein (AKAP), and (4)has 5 different isoforms that differ in WD repeat number. These findingssuggest an important role for lrba in coupling signal transduction andvesicle trafficking to enable polarized secretion and/or membranedeposition of immune effector molecules. This disclosure provides noveltools and methods that can be used to further the understanding of themechanism of CHS and other related diseases as well as general immunecell function.

The cell membrane system not only delimits and protects cell andintracellular organelles, maintaining the essential differences betweenthe cell interior and the environment, but also transports variousmolecules back and forth between the membrane-bound compartments in thecell, and between the cell and the environment through vesicletrafficking processes. These processes are critical for the correctbiological functioning of a eukaryotic cell. A novel gene family, WBW,may play an essential role in vesicle trafficking has been identified ineukaryotic organisms from the very ancient unicellular organismDictyostelium to human, but not in prokaryotes, which have no vesiclesystem (Wang, J. W. et al. Journal of Immunology, 2001,166(7):4586-4595; Kwak, E. et al. Mol. Biol. Cell, 1999,10(12):4429-4439; Adam-Klages, S. et al. Cell, 1996, 86(6):937-947;Barbosa, M. D. et al Nature, 1996, 382(6588):262-265; Nagle, D. L. etal. Nat. Genet., 1996, 14(3):307-311). The WBW proteins all have ahighly conserved long WBW(WDL-BEACH-WD) structure composed of threedomains at their C-termini (Wang, J. W. et al. Journal of Immunology,2001, 166(7):4586-4595). WD domain is present in over two thousandproteins and is thought to be involved in protein-protein interaction(Smith, T. F. et al. Trends Biochem. Sci., 1999, 24(5):181-185). The WDrepeats of FAN bind to NSD motif of TNFR55 to mediate the activation ofthe plasma membrane-bound neutral sphingomyelinase, producing thesecondary messenger ceramide to activate raf-1 and MAP kinases, leadingto cell growth and inflammation responses (Adam-Klages, S. et al. Cell,1996, 86(6):937-947). The function of the BEACH domain is unclear, itpotentially has SH3 and SH2 binding sites and a tyrosine kinasephosphorylation site, and those sites may interact with multiple signaltransduction proteins (Wang, J. W. et al. Journal of Immunology, 2001,166(7):4586-4595). The WDL domain was first described in a previouspublication, and its function also remains unknown (Wang, J. W. et al.Journal of Immunology, 2001, 166(7):4586-4595). However, the WBWstructure is very conserved and the WBW structure of FAN represents mostof its ORF, and thus it is reasonable to propose that the WBW structurehas a similar function to that of FAN. Another interesting question isif WBW proteins are also AKAPs (A kinase anchor protein), as DAKAP550and Neurobeachin have been experimentally proved to be AKAPs, which candirect protein kinase A to discrete intracellular locations, where PKAmay be activated by the secondary messenger cAMP (Han, J. D. et al. J.Biol. Chem., 1997, 272(42):26611-26619; Wang, X. et al J. Neurosci.,2000, 20(23):8551-8565). The subcellular localizations of the WBWproteins are not restricted to the plasma membrane, but are found in theGolgi complex, lysosomes, ER, perinuclear ER and clathrin-coatedendocytosis pits (Wang, J. W. et al. Journal of Immunology, 2001,166(7):4586-4595; Wang, X. et al. J. Neurosci., 2000, 20(23):8551-8565),moreover are associated with microtubules (Faigle, W. et al. J. CellBiol., 1998, 141(5):1121-1134).

In the WBW family chs1/beige gene is the most extensively studied. Themutations of the gene can cause a generalized immunodeficiency in miceand humans with the impairment of NK cells, CTL, and granulocytes andoften cause premature death in humans due to a second disease phasecharacterized by a lymphoproliferative syndrome, probably as a result ofdefective intracellular trafficking of vesicles (Spritz, R. A. et al. J.Clin. Immunol., 1998, 18(2):97-105). For example, the deposition of somemembrane proteins (HLA-DR) and antigen presentation are affected(Faigle, W. et al. J. Cell Biol., 1998, 141(5):1121-1134). FAN has arole in TNF pathway by binding to a cytoplasmic motif upstream of thedeath domain of some TNF family receptors (TNFR55 and CD40)(Adam-Klages, S. et al. Cell, 1996, 86(6):937-947; Segui, B. et al. J.Biol. Chem., 1999, 274(52):37251-37258). FAN knockout or FANdominant-negative form can protect cell from apoptosis mediated by CD40or TNF receptor (Segui, B. et al. J. Clin. Invest., 2001,108(1):143-151; Segui, B. et al. J. Biol. Chem., 1999,274(52):37251-37258). LvsA gene is essential for cytokinesis by possiblyplaying an important role in a membrane-processing pathway (Kwak, E. etal. Mol. Biol. Cell, 1999, 10(12):4429-4439). These studies suggest thatthe WBW proteins may play a role not only in vesicle trafficking, butalso in some important cell processes like apoptosis and cell cycle.

However, the exact molecular mechanism of vesicle trafficking for theWBW proteins remains largely unclear. The mouse lrba (LPS-responsivebeige-like PKA anchor gene) has its three isoforms, which differ atC-termini and have tissue-specific and development stage-specificexpression pattern. LRBA gene is LPS inducible and can physicallyassociate with various vesicular compartments in cells (Wang, J. W. etal. Journal of Immunology, 2001, 166(7):4586-4695). Described herein isthe cloning, genomic structure and promoter analysis of the human lrbagene and its five isoforms. Its genomic locus consists of 58 exons and57 introns, spinning over 700 K bps. Three isoforms (α, β, δ) differ atBEACH domain and WD repeats at their C-termini. The fourth isoform(γ)has a YLLLQ insertion sequence. The mRNA of the fifth isoform (δ) hastwo ORFs and a potential IRES for the translation of the second ORF. Inthe promoter region, there are four E2F binding sites and a CpG island,and surprisingly a potential p53 binding site was found in the promoter,suggesting that lrba gene may be involved in p53 mediated apoptosis orcell arrest, and E2F regulated cell cycle progress, and is regulateddevelopmentally by CpG island. These results show that the Lrba gene ishighly regulated at both the transcriptional and translational level,indicating that lrba gene may have a critical role in the life of thecell.

All patents, patent applications, provisional applications,publications, and nucleic acid and amino acid sequences associated withthe GenBank accession numbers referred to or cited herein areincorporated by reference in their entirety, including all figures andtables, to the extent they are not inconsistent with the explicitteachings of this specification.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication.

1. A method for inhibiting the growth of a tumor characterized byoverexpression of the LPS-responsive CHS1/beige-like anchor gene (lrba)in a mammal, comprising directly administering an effective amount of aninterfering RNA molecule to the tumor, wherein the interfering RNA isspecific for lrba mRNA within the mammal and reduces lrba expressionwithin the tumor, and wherein the interfering RNA inhibits growth of thetumor in the mammal.
 2. The method according to claim 1, wherein theinterfering RNA is single-stranded RNA selected from the groupconsisting of SEQ ID NO. 177, SEQ ID NO. 178, SEQ ID NO. 179, and SEQ IDNO.
 180. 3. The method according to claim 1, wherein the interfering RNAis double-stranded RNA comprising SEQ ID NO. 177, SEQ ID NO. 178, SEQ IDNO. 179, or SEQ ID NO.
 180. 4. The method of claim 1, wherein the mammalis a human.
 5. The method according to claim 1, wherein the tumor is ofa cancer type selected from the group consisting of breast, prostate,melanoma, cervical cancer, adenocarcinoma, colorectal cancer, and lungcarcinoma.
 6. The method according to claim 1, wherein the tumor is of acancer type selected from breast cancer or prostate cancer.
 7. A methodfor inhibiting the growth of mammalian cancer cells characterized byoverexpression of the LPS-responsive CHS1/beige-like anchor gene (lrba),comprising directly administering an effective amount of an interferingRNA molecule to the cancer cells in vitro or in vivo, wherein theinterfering RNA is specific for lrba mRNA within the cancer cells andreduces lrba expression within the cancer cells, and wherein theinterfering RNA inhibits growth of the cancer cells.
 8. The methodaccording to claim 7, wherein the cancer cells are of a cancer typeselected from the group consisting of breast, prostate, melanoma,cervical cancer, adenocarcinoma, colorectal cancer, and lung carcinoma.9. The method according to claim 7, wherein the cancer cells are of acancer type selected from breast cancer or prostate cancer.
 10. Themethod according to claim 7, wherein the cancer cells are human cells.11. The method according to claim 7, wherein said administering iscarried out in vivo.